Methods and compositions for treating mitochondrial disease or disorders and heteroplasmy

ABSTRACT

The present invention provides methods and compositions for generation of mitochondria replaced cells (MirC), and therapeutic methods for using such compositions for treating a subject having an age-related disease or syndrome, mitochondrial disease or disorder, or otherwise in need of mitochondrial replacement. Also provided are methods and compositions for producing a recipient cell having a mitochondrial disease or disorder, as well as methods and compositions for producing or enhancing production of an inducible pluripotent stem cell (iPSC). In addition, methods and compositions to enhance mitochondrial transfer are also included.

This application claims the benefit of U.S. Provisional Application No.62/718,891, filed Aug. 14, 2018, U.S. Provisional Application No.62/731,731 filed Sep. 14, 2018, and U.S. Provisional Application No.62/817,987 filed Mar. 13, 2019, which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 13, 2019, isnamed 14595-001-999_SL.txt and is 12,905 bytes in size.

Field of the Invention

The present invention provides a composition of cells with reducedmitochondrial DNA and/or replacement of mitochondrial DNA, methods fortheir production, and methods for treating various diseases associatedwith genetic or age-related mitochondrial dysfunctions.

Background of the Invention

Mitochondria play a major and critical role in cellular homeostasis, andare involved in a diverse range of disease processes. They participatein intracellular signaling, apoptosis and perform numerous biochemicaltasks, such as pyruvate oxidation, the Krebs cycle, and metabolism ofamino acids, fatty acids, nucleotides and steroids. One crucial task istheir role in cellular energy metabolism. This includes β-oxidation offatty acids and production of ATP by means of the electron-transportchain and the oxidative-phosphorylation system. The mitochondrialrespiratory chain consists of five multi-subunit protein complexesembedded in the inner membrane, comprising: complex I (NADH-ubiquinoneoxidoreductase), complex II (succinate-ubiquinone oxidoreductase),complex III (ubiquinol-ferricytochrome c oxidoreductase), complex IV(cytochrome c oxidoreductase), and complex V (FIFO ATPase).

The mammalian mitochondrial genome is a small, circular, double-strandedmolecule containing 37 genes, including 13 protein-encoding genes, 22transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Of these,24 (22 tRNAs and two rRNAs) are needed for mitochondrial DNAtranslation, and 13 encode subunits of the respiratory chain complexes.In addition, nuclear DNA (nDNA) encodes most of the approximately 900gene products in the mitochondria.

Mitochondrial disease or disorders are a clinically heterogeneous groupof disorders that are characterized by dysfunctional mitochondria.Disease onset can occur at any age and can manifest with a wide range ofclinical symptoms. Mitochondrial disease or disorders can involve anyorgan or tissue, characteristically involve multiple systems, typicallyaffecting organs that are highly dependent on aerobic metabolism, andare often relentlessly progressive with high morbidity and mortality.Mitochondrial disease or disorders are the most common group ofinherited metabolic disorders and are among the most common forms ofinherited neurological disorders.

Mitochondrial disease or disorders can be caused by mutations in genesin the nuclear DNA (nDNA) and/or mitochondrial DNA (mtDNA) that encodestructural mitochondrial proteins or proteins involved in mitochondrialfunction. While some mitochondrial disorders only affect a single organ(e.g., the eye in Leber hereditary optic neuropathy [LHON]), manyinvolve multiple organ systems and often present with prominentneurologic and myopathic features. Even though tissues with high energydemand, such as brain, muscle, and eye, are more frequently involved,patients' phenotype can be extremely varied and heterogeneous. Thisvariation is due in part because of several factors, such as, the dualgenetic control (nDNA and mtDNA), level of heteroplasmy (percentage ofmutated DNA in single cells and tissues), tissue energy demand, maternalinheritance, and mitotic segregation.

Many patients with a mitochondrial disease or disorder have a mixture ofmutated and wild-type mtDNA (known as heteroplasmy); the proportion ofmutated and wild-type mtDNA is a key factor that determines whether acell expresses a biochemical defect. The majority of pathogenetic mtDNAmutations are heteroplasmic, with a mixture of mutated and wild-typemtDNA inside an individual cell. High levels of heteroplasmy refer tocells with high levels of mutant mtDNA and low levels of wild-typemtDNA, whereas low levels of heteroplasmy refer to cells with low levelsof mutant mtDNA and high levels of wild-type mtDNA. Studies in singlecells from patients with mitochondrial disease or disorders have shownthat the level of mutated and wild-type mtDNA is very important fordetermining the cellular phenotype. For example, cells becomerespiratory deficient if they contain high levels of mutated mtDNA andlow levels of wild-type mtDNA (that is, high levels of heteroplasmy).The threshold at which this deficiency occurs depends on the precisemutation and the cell type. Typically, high percentage levels of mutatedmtDNA (>50%) are required to result in cellular defects, but some mtDNAmutations only generate a deficiency if present at very high levels(typically mt tRNA mutations) and others (such as single, large-scalemtDNA deletions) produce a deficiency when there is ˜60% deleted mtDNA.For example, in individuals harboring the m.8993T>G pathogenic variant,higher percentage levels of mutated mtDNA are seen in those presentingwith Leigh syndrome than in those presenting with neurogenic weaknesswith ataxia and retinitis pigmentosa (NARP). In addition, clinicalphenotypes in MELAS and MERRF correlate with heteroplasmy (see, e.g.,Chinnery, P. F., et al., Brain 120 (Pt 10), 1713-1721 (1997)).

Advances in next-generation sequencing technology have revealed manymutations that cause mitochondrial disease or disorders. In addition,investigations into other organisms, such as C. elegans, have revealedsome of the proteins involved in heteroplasmy. For example, a recentstudy using C. elegans demonstrated that mitochondrial unfolded proteinresponse (UPRmt) functions to maintain the heteroplasmy and propagatemutated mtDNA following a disturbance of the original mtDNA (see, e.g.,Lin, Y. F. et al. Nature 533, 416-419, doi:10.1038/nature17989 (2016)).However, the mechanism related to heteroplasmy maintenance andpropagation in mammalian cells remains unknown.

The management and treatment of patients with mitochondrial disease ordisorders remains challenging. For the vast majority of patients, thecondition is relentlessly progressive leading to considerable morbidityand, in those most severely affected, death. The classical method toremove endogenous mtDNA involves long term treatment of cells with lowconcentrations of ethidium bromide (EtBr), a known carcinogen andteratogen, limiting its application for therapeutic purposes. Inaddition to the potential for unwanted side effects, the EtBr protocolcan take several months, which further limits its clinical use.Moreover, mitochondrial transfer protocols generally involve a completedepletion of endogenous mtDNA, termed rho (ρ) 0 cells, before transferof exogenous mitochondria. This complete depletion of mtDNA severelyhinders the ability of a cell to ingest exogenous mitochondria.

Other mitochondrial transfer protocols have attempted to addmitochondria without depletion of endogenous mtDNA, but this approachhas been found to be inefficient or harmful to a cell. For example,mitochondrial transfer using simple coincubation has been reported to beineffective and not equally efficient among different cell types.Additional techniques to transfer have involved injection using invasiveinstruments, which caused harm to the recipient cell, or other invasiveinstruments, such as nanoblades, but all were less efficient thancoincubation (Caicedo et al, Stem Cells International, (2017), vol.2017, Article ID 7610414, 23 pages).

Accordingly, current methods of mitochondrial transfer are not onlyimpractical for the clinical setting, but they are also inefficient,harmful to recipient cells and/or time intensive. Thus, there is asignificant unmet need to develop improved methods for mitochondrialtransfer that can be optionally used in the treatment of a subjecthaving or suspected having mitochondrial disease or disorders, anddiseases or disorders associated with impaired or dysfunctionalmitochondria, as well as improved models for studying mitochondrialdisease or disorders.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a method of generating a mitochondriareplaced cell, comprising: (a) contacting a recipient cell with an agentthat reduces endogenous mtDNA copy number; (b) incubating the recipientcell for a sufficient period of time for the agent to partially reducethe endogenous mtDNA copy number in the recipient cell; and (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA has been partially reduced, and (2) exogenousmitochondria from a healthy donor, for a sufficient period of time tonon-invasively transfer exogenous mitochondria into the recipient cell,thereby generating a mitochondria replaced cell.

In another aspect, provided herein is a method of treating a subject inneed of mitochondrial replacement, comprising (a) generating amitochondria replaced cell ex vivo or in vitro, comprising the steps of(i) contacting a recipient cell with an agent that reduces mtDNA copynumber (ii) incubating the recipient cell for a sufficient period oftime for the agent to partially reduce the mtDNA copy number in therecipient cell; and (iii) co-incubating (1) the recipient cell from step(ii) in which the endogenous mtDNA has been partially reduced, and (2)exogenous mitochondria from a healthy donor, for a sufficient period oftime to non-invasively transfer exogenous mitochondria into therecipient cell, thereby generating a mitochondria replaced cell; and (b)administering a therapeutically effective amount of the mitochondriareplaced recipient cell from step (a) to the subject in need ofmitochondrial replacement.

In yet another aspect, provided herein is a method of treating a subjecthaving or suspected of having an age-related disease, the methodcomprising: (a) generating a mitochondria replaced cell ex vivo or invitro, comprising the steps of: (i) contacting a recipient cell with anagent that reduces mtDNA copy number; (ii) incubating the recipient cellfor a sufficient period of time for the agent to partially reduce themtDNA copy number in the recipient cell; and (iii) co-incubating (1) therecipient cell from step (ii) in which the endogenous mtDNA has beenpartially reduced, and (2) exogenous mitochondria from a healthy donor,for a sufficient period of time to non-invasively transfer exogenousmitochondria into the recipient cell, thereby generating a mitochondriareplaced cell; and (b) administering a therapeutically effective amountof the mitochondria replaced recipient cell from step (a) to the subjecthaving or suspected of having an age-related disease.

In a further aspect, provided herein is a method of treating a subjecthaving or suspected of having a mitochondrial disease or disorder, themethod comprising: (a) generating a mitochondria replaced recipient cellex vivo or in vitro, comprising the steps of: (i) contacting a recipientcell with an agent that reduces mtDNA copy number; (ii) incubating therecipient cell for a sufficient period of time for the agent topartially reduce the mtDNA copy number in the recipient cell; and (iii)co-incubating (1) the recipient cell from step (ii) in which theendogenous mtDNA has been partially reduced, and (2) exogenousmitochondria from a healthy donor, for a sufficient period of time tonon-invasively transfer exogenous mitochondria into the recipient cell,thereby generating a mitochondria replaced cell; and (b) administering atherapeutically effective amount of the mitochondria replaced recipientcell from step (a) to the subject having or suspected of having amitochondrial disease or disorder.

In some embodiments of the methods provided herein, the exogenousmitochondria is a functional mitochondria. In certain embodiments, theexogenous mitochondria comprises wild-type mtDNA. In specificembodiments, the exogenous mitochondria is isolated mitochondria. Infurther embodiments, the isolated mitochondria is an intactmitochondria. In some embodiments, the exogenous mitochondria isallogeneic.

Also provided herein is a method of generating a mitochondria replacedcell, comprising (a) contacting a recipient cell with an agent thatreduces endogenous mtDNA copy number; (b) incubating the recipient cellfor a sufficient period of time for the agent to partially reduce theendogenous mtDNA copy number in the recipient cell; and (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA has been partially reduced, and (2) exogenous mtDNAfrom a healthy donor, for a sufficient period of time to non-invasivelytransfer exogenous mtDNA into the recipient cell, thereby generating amitochondria replaced cell.

The disclosure also provides a method of treating a subject in need ofmitochondrial replacement, comprising (a) generating a mitochondriareplaced cell ex vivo or in vitro, comprising the steps of: (i)contacting a recipient cell with an agent that reduces mtDNA copynumber, (ii) incubating the recipient cell for a sufficient period oftime for the agent to partially reduce the mtDNA copy number in therecipient cell; and (iii) co-incubating (1) the recipient cell from step(ii) in which the endogenous mtDNA has been partially reduced, and (2)exogenous mtDNA from a healthy donor, for a sufficient period of time tonon-invasively transfer exogenous mtDNA into the recipient cell, therebygenerating a mitochondria replaced cell; and (b) administering atherapeutically effective amount of the mitochondria replaced recipientcell from step (a) to the subject in need of mitochondrial replacement.

In another aspect, provided herein is a method of treating a subjecthaving or suspected of having an age-related disease, the methodcomprising: (a) generating a mitochondria replaced cell ex vivo or invitro, comprising the steps of: (i) contacting a recipient cell with anagent that reduces mtDNA copy number; (ii) incubating the recipient cellfor a sufficient period of time for the agent to partially reduce themtDNA copy number in the recipient cell; and (iii) co-incubating (1) therecipient cell from step (ii) in which the endogenous mtDNA has beenpartially reduced, and (2) exogenous mtDNA from a healthy donor, for asufficient period of time to non-invasively transfer exogenous mtDNAinto the recipient cell, thereby generating a mitochondria replacedcell; and (b) administering a therapeutically effective amount of themitochondria replaced recipient cell from step (a) to the subject havingor suspected of having an age-related disease.

In yet another aspect, provided herein is a method of treating a subjecthaving or suspected of having a mitochondrial disease or disorder, themethod comprising: (a) generating a mitochondria replaced recipient cellex vivo or in vitro, comprising the steps of: (i) contacting a recipientcell with an agent that reduces mtDNA copy number; (ii) incubating therecipient cell for a sufficient period of time for the agent topartially reduce the mtDNA copy number in the recipient cell; and (iii)co-incubating (1) the recipient cell from step (ii) in which theendogenous mtDNA has been partially reduced, and (2) exogenous mtDNAfrom a healthy donor, for a sufficient period of time to non-invasivelytransfer exogenous mtDNA into the recipient cell, thereby generating amitochondria replaced cell; and (b) administering a therapeuticallyeffective amount of the mitochondria replaced recipient cell from step(a) to the subject having or suspected of having a mitochondrial diseaseor disorder.

In certain embodiments of the methods provided herein, the agent thatreduces endogenous mtDNA copy number is selected from the groupconsisting of a polynucleotide encoding a fusion protein comprising amitochondrial-targeted sequence (MTS) and an endonuclease, apolynucleotide encoding an endonuclease, and a small molecule. In someembodiments, the small molecule is a nucleoside reverse transcriptaseinhibitor (NRTI). In other embodiments, the polynucleotide is comprisedof messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA). Infurther embodiments, the recipient cell transiently expresses the fusionprotein. In yet further embodiments, the endonuclease is selected fromthe group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zincfinger nuclease (ZFN), and transcription activator-like effectornuclease (TALEN). In some embodiments, the MTS targets a mitochondrialmatrix protein. In specific embodiments, the mitochondrial matrixprotein is selected from the group consisting of cytochrome c oxidasesubunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidasesubunit X.

In some embodiments of the methods provided herein, the agent thatreduces endogenous mtDNA copy number reduces about 5% to about 99% ofthe endogenous mtDNA copy number. In certain embodiments, the agent thatreduces endogenous mtDNA copy number reduces about 30% to about 70% ofthe endogenous mtDNA copy number. In further embodiments, the agent thatreduces endogenous mtDNA copy number reduces about 50% to about 95% ofthe endogenous mtDNA copy number. In yet further embodiments, the agentthat reduces endogenous mtDNA copy number reduces about 60% to about 90%of the endogenous mtDNA copy number. In some embodiments, the agent thatreduces endogenous mtDNA copy number reduces mitochondrial mass.

Also provided herein is a method of generating a mitochondria replacedcell, comprising: (a) contacting a recipient cell with an agent thatreduces mitochondrial function; (b) incubating the recipient cell for asufficient period of time for the agent to partially reduce theendogenous mitochondrial function in the recipient cell; and (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mitochondrial function has been partially reduced, and (2)exogenous mitochondria from a healthy donor, for a sufficient period oftime to non-invasively transfer exogenous mitochondria into therecipient cell, thereby generating a mitochondria replaced cell.

The present disclosure also provides a method of generating amitochondria replaced cell, comprising: (a) contacting a recipient cellwith an agent that reduces mitochondrial function; (b) incubating therecipient cell for a sufficient period of time for the agent topartially reduce the endogenous mitochondrial function in the recipientcell; and (c) co-incubating (1) the recipient cell from step (b) inwhich the endogenous mitochondrial function has been partially reduced,and (2) exogenous mtDNA from a healthy donor, for a sufficient period oftime to non-invasively transfer exogenous mtDNA into the recipient cell,thereby generating a mitochondria replaced cell.

In some embodiments of the methods provided herein, the agent thatreduces mitochondrial function transiently reduces endogenousmitochondrial function. In other embodiments, the agent that reducesmitochondrial function permanently reduces endogenous mitochondrialfunction.

In certain embodiments of the methods provided herein, the subject inneed of mitochondrial replacement has a dysfunctional mitochondria; adisease selected from the group consisting of an age-related disease, amitochondrial disease or disorder, a neurodegenerative disease, aretinal disease, diabetes, a hearing disorder, a genetic disease; or acombination thereof. In some embodiments, the neurodegenerative diseaseis selected from the group consisting of amyotrophic lateral sclerosis(ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease,Friedreich's ataxia, Charcot Marie Tooth disease and leukodystrophy. Inspecific embodiments, the retinal disease is selected from the groupconsisting of age-related macular degeneration, macular edema andglaucoma.

In some embodiments of the methods provided herein, the age-relateddisease is selected from the group consisting of an autoimmune disease,a metabolic disease, a genetic disease, cancer, a neurodegenerativedisease, and immunosenescence. In certain embodiments of the methodsprovided herein, the metabolic disease is diabetes. In furtherembodiments, the neurodegenerative disease is Alzheimer's disease, orParkinson's disease. In yet further embodiments, the genetic disease isselected from the group consisting of Hutchinson-Gilford ProgeriaSyndrome, Werner Syndrome, and Huntington's disease.

In certain embodiments of the methods provided herein, the mitochondrialdisease or disorder is caused by mitochondrial DNA abnormalities,nuclear DNA abnormalities, or both. In specific embodiments, themitochondrial disease or disorder caused by mitochondrial DNAabnormalities is selected from the group consisting of chronicprogressive external ophthalmoplegia (CPEO), Pearson syndrome,Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrialdiabetes, Leber hereditary optic neuropathy (LHON), LHON-plus,neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP),maternally-inherited Leigh syndrome (MILS), mitochondrialencephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS),myoclonic epilepsy and ragged-red fiber disease (MERRF), familialbilateral striatal necrosis/striatonigral degeneration (FBSN), Luftdisease, aminoglycoside-induced Deafness (AID), and multiple deletionsof mitochondrial DNA syndrome. In yet other specific embodiments, themitochondrial disease or disorder caused by nuclear DNA abnormalities isselected from the group consisting of Mitochondrial DNA depletionsyndrome-4A, mitochondrial recessive ataxia syndrome (MIRAS),mitochondrial neurogastrointestinal encephalomyopathy (MNGIE),mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma(POLG)-related disorders, sensory ataxia neuropathy dysarthriaophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinalcord involvement and lactate elevation (LB SL), co-enzyme Q10deficiency, Leigh syndrome, mitochondrial complex abnormalities,fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC)deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenasecomplex deficiency (PDHC), pyruvate carboxylase deficiency (PCD),carnitine palmitoyltransferase I (CPT I) deficiency, carnitinepalmitoyltransferase II (CPT II) deficiency, carnitine-acyl-carnitine(CACT) deficiency, autosomal dominant-/autosomal recessive-progressiveexternal ophthalmoplegia (ad-/ar-PEO), infantile onset spinal cerebellaratrophy (IOSCA), mitochondrial myopathy (MM) spinal muscular atrophy(SMA), growth retardation, aminoaciduria, cholestasis, iron overload,early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

In some embodiments of the methods provided herein the endogenous mtDNAencodes for a dysfunctional mitochondria. In specific embodiments, theendogenous mtDNA comprises mutant mtDNA. In other embodiments, theendogenous mtDNA in the recipient cell comprises wild-type mtDNA. In yetfurther embodiments, the endogenous mtDNA comprises mtDNA associatedwith a mitochondrial disease or disorder. In some embodiments, theendogenous mtDNA is heteroplasmic. In specific embodiments, therecipient cell has endogenous mitochondria that is dysfunctional.

In certain embodiments of the methods provided herein, the mitochondriareplaced cell has a total mtDNA copy number no greater than about 1.1fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, ormore, relative to the total mtDNA copy number of the recipient cellprior to contacting with the agent that reduces endogenous mtDNA copynumber.

In some embodiments, the recipient cell is an animal cell or a plantcell. In certain embodiments, the animal cell is a mammalian cell. Inspecific embodiments, the recipient cell is a somatic cell. In otherembodiments, the recipient cell is a bone marrow cell. In someembodiments, the bone marrow cell is a hematopoietic stem cell (HSC), ora mesenchymal stem cell (MSC). In other embodiments, the recipient cellis a cancer cell. In further embodiments, the recipient cell is aprimary cell. In yet further embodiments, the recipient cell is animmune cell. In specific embodiments, the immune cells is selected fromthe group consisting of a T cell, a phagocyte, a microglial cell, and amacrophage. In further embodiments, the T cell is a CD4+ T cells. Inother embodiments, the T cell is a CD8+ T cells. In certain embodiments,the T cell is a chimeric antigen receptor (CAR) T cell.

In another embodiment of the methods provided herein, the exogenousmitochondria and/or exogenous mtDNA is stable. In some embodiments, theexogenous mtDNA alters heteroplasmy in the recipient cell.

In some aspects of the methods provided herein, the method furthercomprises delivering a small molecule, a peptide, or a protein.

The disclosure also provides methods provided herein, further comprisingcontacting the recipient cell with a second active agent prior toco-incubating the recipient cell with exogenous mitochondria and/orexogenous mtDNA. In certain embodiments, the second active agent isselected from the group consisting of large molecules, small molecules,or cell therapies, and the second active agent is optionally selectedfrom the group consisting of rapamycin, NR (Nicotinamide Riboside),bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide(MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thiocticacid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis. In some embodiments, the activator ofendocytosis is a modulator of cellular metabolism. In specificembodiments, the modulator of cellular metabolism comprises nutrientstarvation, a chemical inhibitor, or a small molecule. In yet furtherembodiments, the chemical inhibitor or the small molecule is an mTORinhibitor. In even further embodiments, the mTOR inhibitor comprisesrapamycin or a derivative thereof.

The disclosure also provides a composition comprising one or moremitochondria replaced cells obtained by the method of: (a) contacting arecipient cell with an agent that reduces endogenous mtDNA copy number;(b) incubating the recipient cell for a sufficient period of time forthe agent to partially reduce the endogenous mtDNA copy number in therecipient cell; and (c) co-incubating (1) the recipient cell from step(b) in which the endogenous mtDNA has been partially reduced, and (2)exogenous mitochondria from a healthy donor, for a sufficient period oftime to non-invasively transfer exogenous mitochondria into therecipient cell, thereby generating a mitochondria replaced cell, whereinsaid mitochondria replaced cell comprises greater than 5% of exogenousmtDNA.

The disclosure further provides a composition of one or moremitochondria replaced cells obtained by the method of (a) contacting arecipient cell with an agent that reduces endogenous mtDNA copy number(b) incubating the recipient cell for a sufficient period of time forthe agent to partially reduce the endogenous mtDNA copy number in therecipient cell; and (c) co-incubating (1) the recipient cell from step(b) in which the endogenous mtDNA has been partially reduced, and (2)exogenous mtDNA from healthy donor, for a sufficient period of time tonon-invasively transfer exogenous mtDNA into the recipient cell, therebygenerating a mitochondria replaced cell, wherein said mitochondriareplaced cell comprises greater than 5% of exogenous mtDNA. In someembodiments of the compositions provided herein, the one or moremitochondria replaced cells comprise a total mtDNA copy number nogreater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4fold, about 1.5 fold, or more, relative to the total mtDNA copy numberof the recipient cell prior to contacting with the agent that reducesendogenous mtDNA copy number.

In another aspect, provided herein is a composition for use in a methodof generating one or more mitochondria replaced cells comprising anagent that reduces endogenous mtDNA copy number, and a second activeagent. In some embodiments, the composition further comprising one ormore recipient cells, or a combination thereof. In certain embodiments,the composition further comprising exogenous mtDNA exogenous mtDNAand/or exogenous mitochondria.

In certain embodiments of the compositions provided herein, the agentthat reduces endogenous mtDNA copy number is a small molecule or afusion protein. In some embodiments, the small molecule is a nucleosidereverse transcriptase inhibitor (NRTI). In other embodiments, the fusionprotein comprises an endonuclease that cleaves mtDNA and a mitochondrialtarget sequence (MTS). In some embodiments, the endonuclease cleaveswild-type mtDNA. In specific embodiments, the endonuclease is selectedfrom the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9,zinc finger nuclease (ZFN), and transcription activator-like effectornuclease (TALEN). In some embodiments, the MTS targets a mitochondrialmatrix protein. In further embodiments, the mitochondrial matrix proteinis cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII,and cytochrome c oxidase subunit X. In specific embodiments, the fusionprotein is transiently expressed.

In some embodiments of the compositions provided herein, the reductionof endogenous mtDNA copy number is a partial reduction. In certainembodiments, the partial reduction is a reduction of about 5% to about99% of endogenous mtDNA. In specific embodiments, the partial reductionis a reduction of about 50% to about 95% of the endogenous mtDNA copynumber. In further embodiments, the partial reduction is a reduction ofabout 60% to about 90% of the endogenous mtDNA copy number.

The disclosure also provides a composition comprising one or moremitochondria replaced cells obtained by the method of: (a) contacting arecipient cell with an agent that reduces mitochondrial function; (b)incubating the recipient cell for a sufficient period of time for theagent to partially reduce endogenous mitochondrial function in therecipient cell; and (c) co-incubating (1) the recipient cell from step(b) in which the endogenous mitochondrial function has been partiallyreduced, and (2) exogenous mitochondria from a healthy donor, for asufficient period of time to non-invasively transfer exogenousmitochondria into the recipient cell, thereby generating a mitochondriareplaced cell, wherein said mitochondria replaced cell comprises greaterthan 5% of exogenous mtDNA.

In another aspect, provided herein is a composition of one or moremitochondria replaced cells obtained by the method of: (a) contacting arecipient cell with an agent that reduces mitochondrial function; (b)incubating the recipient cell for a sufficient period of time for theagent to partially reduce endogenous mitochondrial function in therecipient cell; and (c) co-incubating (1) the recipient cell from step(b) in which the endogenous mitochondrial function has been partiallyreduced, and (2) exogenous mtDNA from healthy donor, for a sufficientperiod of time to non-invasively transfer exogenous mtDNA into therecipient cell, thereby generating a mitochondria replaced cell, whereinsaid mitochondria replaced cell comprises greater than 5% of exogenousmtDNA. In some embodiments, the one or more mitochondria replaced cellscomprise a total mtDNA copy number no greater than about 1.1 fold, about1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more,relative to the total mtDNA copy number of the recipient cell prior tocontacting with the agent that reduces endogenous mtDNA copy number.

The disclosure also provides a composition for use in a method ofgenerating one or more mitochondria replaced cells comprising an agentthat reduces mitochondrial function, and a second active agent. In someembodiments, the composition further comprises an exogenousmitochondria, one or more recipient cells, or a combination thereof. Inyet further embodiments, the composition further comprises exogenousmtDNA.

In some embodiments of the compositions provided herein, the one or moremitochondria replaced cells comprise wild-type exogenous mtDNA.

Also provided herein are compositions further comprising a second activeagent. In some embodiments, the second active agent is selected from thegroup consisting of large molecules, small molecules, or cell therapies,and the second active agent is optionally selected from the groupconsisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate,idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131),omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid,A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis. In specific embodiments, the activator ofendocytosis is an activator of a clathrin-independent endocytosispathway. In some embodiments, the activator of endocytosis is anactivator of a clathrin-independent endocytosis pathway. In furtherembodiments, the clathrin-independent endocytosis pathway is selectedfrom the group consisting of a CLIC/GEEC endocytic pathway,Arf6-dependent endocytosis, flotillin-dependent endocytosis,macropinocytosis, circular doral ruffles, phagocytosis, andtrans-endocytosis. In yet further embodiments, the clathrin-independentendocytosis pathway is macropinocytosis. In specific embodiments, theactivator of endocytosis comprises nutrient stress, and/or an mTORinhibitor. In some embodiments, the mTOR inhibitor comprises rapamycinor a derivative thereof.

In certain embodiments, the disclosure further provides a compositionwhere the total mtDNA copy number of the one or more mitochondriareplaced cells comprises greater than 5% of exogenous mtDNA. In someembodiments, the total mtDNA copy number of the one or more mitochondriareplaced cells comprises greater than 30% of exogenous mtDNA. Inspecific embodiments, the total mtDNA copy number of the one or moremitochondria replaced cells comprises greater than 50% of exogenousmtDNA. In further embodiments, the total mtDNA copy number of the one ormore mitochondria replaced cells comprises greater than 75% of exogenousmtDNA.

In some embodiments of the compositions provided herein, the exogenousmitochondria is isolated mitochondria. In specific embodiments, theisolated mitochondria is intact. In some embodiments, the exogenousmitochondria and/or exogenous mtDNA is allogeneic. In specificembodiments, the exogenous mitochondria further comprises exogenousmtDNA.

In certain embodiments of the compositions provided herein, the one ormore cells are animal cells or plant cells. In some embodiments, theanimal cells are mammalian cells. In specific embodiments, the cells aresomatic cells. In further embodiments, the somatic cells are epithelialcells. In yet further embodiments, the epithelial cells are thymicepithelial cells (TECs). In other embodiments, the somatic cells areimmune cells. In certain embodiments, the immune cells are T cells. Inspecific embodiments, the T cells are CD4+ T cells. In otherembodiments, the T cells are CD8+ T cells. In some embodiments, the Tcells are chimeric antigen receptor (CAR) T cells. In other embodiments,the immune cells are phagocytic cells. In certain embodiments, the oneor more mitochondria replaced cells are bone marrow cells. In specificembodiments, the bone marrow cells are a hematopoietic stem cell (HSC),or a mesenchymal stem cell (MSC).

In some embodiments of the compositions provided herein, the one or moremitochondria replaced cells are more viable than an isogenic cell havinghomoplasmic endogenous mtDNA. In other embodiments, the one or moremitochondria replaced cells are efficacious in killing a cancer cell,treating an age-related disease, treating a mitochondrial disease ordisorder, treating a neurodegenerative disease, treating diabetes, or agenetic disease.

In certain embodiments of the compositions provided herein, thecomposition further comprises a small molecule, a peptide, or a protein.

Also provided herein is a composition for use in delaying senescenceand/or extending lifespan in a cell comprising: (a) a senescent or nearsenescent cell having endogenous mitochondria; (b) isolated exogenousmitochondria from a non-senescent cell; and (c) an agent that reducesendogenous mtDNA copy number. In some embodiments the agent is a fusionprotein. In certain embodiments, the fusion protein comprises anendonuclease that cleaves mtDNA and a mitochondrial target sequence(MTS). In specific embodiments, the endonuclease cleaves wild-typemtDNA. In some embodiments, the endonuclease is selected from the groupconsisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc fingernuclease (ZFN), and transcription activator-like effector nuclease(TALEN). In further embodiments, the MTS targets a mitochondrial matrixprotein. In yet further embodiments, the mitochondrial matrix protein isselected from the group consisting of cytochrome c oxidase subunit IV,cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X.In certain embodiments, the fusion protein is transiently expressed insaid senescent or near senescent cell.

The disclosure further provides a composition for use in delayingsenescence and/or extending lifespan in a cell comprising: (a) asenescent or near senescent cell having endogenous mitochondria; (b)isolated exogenous mitochondria from a non-senescent cell; and (c) anagent that reduces mitochondrial function. In some embodiments, theagent that reduces mitochondrial function transiently reduces endogenousmitochondrial function. In other embodiments, the agent that reducesmitochondrial function permanently reduces endogenous mitochondrialfunction. In some embodiments, the exogenous mitochondria from thenon-senescent cell has enhanced function relative to the endogenousmitochondria.

In some embodiments, the composition for use in delaying senescenceand/or extending lifespan in a cell further comprises a second activeagent. In specific embodiments, the second active agent is selected fromthe group consisting of large molecules, small molecules, or celltherapies, and the second active agent is optionally selected from thegroup consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate,idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131),omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid,A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis. In some embodiments, the activator ofendocytosis is an activator of a clathrin-independent endocytosispathway. In specific embodiments, the clathrin-independent endocytosispathway is selected from the group consisting of a CLIC/GEEC endocyticpathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis,macropinocytosis, circular doral ruffles, phagocytosis, andtrans-endocytosis. In further embodiments, the clathrin-independentendocytosis pathway is macropinocytosis. In some embodiments, saidactivator of endocytosis comprises nutrient stress, and/or an mTORinhibitor. In certain embodiments, said mTOR inhibitor comprisesrapamycin or a derivative thereof.

In another aspect, the disclosure also provides a pharmaceuticalcomposition comprising an isolated population of mitochondria replacedcells having an exogenous mitochondria from a healthy donor, wherein thecells are obtained by any of the methods provided herein for obtaining amitochondrial replaced cell. In yet another aspect, the disclosureprovides a pharmaceutical composition comprising an isolated populationof mitochondria replaced cells having an exogenous mtDNA from a healthydonor, wherein the cells are obtained by any of the methods providedherein for obtaining a mitochondrial replaced cell. In some embodiments,the pharmaceutical composition comprising an isolated population ofmitochondria replaced cells having an exogenous mtDNA from a healthydonor further comprises exogenous mitochondria.

For example, in some embodiments, a pharmaceutical compositioncomprising an exogenous mitochondria from a healthy donor are obtainedby a method of generating a mitochondrial replaced cell that includes(a) contacting a recipient cell with an agent that reduces endogenousmtDNA copy number; (b) incubating the recipient cell for a sufficientperiod of time for the agent to partially reduce the endogenous mtDNAcopy number in the recipient cell; and (c) co-incubating (1) therecipient cell from step (b) in which the endogenous mtDNA has beenpartially reduced, and (2) exogenous mitochondria from a healthy donor,for a sufficient period of time to non-invasively transfer exogenousmitochondria into the recipient cell, thereby generating a mitochondriareplaced cell. In certain embodiments, the cells are obtained by amethod comprising (a) contacting a recipient cell with an agent thatreduces mitochondrial function; (b) incubating the recipient cell for asufficient period of time for the agent to partially reduce theendogenous mitochondrial function in the recipient cell; and (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mitochondrial function has been partially reduced, and (2)exogenous mitochondria from a healthy donor, for a sufficient period oftime to non-invasively transfer exogenous mitochondria into therecipient cell, thereby generating a mitochondria replaced cell.

In other embodiments, the cells are obtained by a method comprising (a)contacting a recipient cell with an agent that reduces endogenous mtDNAcopy number; (b) incubating the recipient cell for a sufficient periodof time for the agent to partially reduce the endogenous mtDNA copynumber in the recipient cell; and (c) co-incubating (1) the recipientcell from step (b) in which the endogenous mtDNA has been partiallyreduced, and (2) exogenous mtDNA from a healthy donor, for a sufficientperiod of time to non-invasively transfer exogenous mtDNA into therecipient cell, thereby generating a mitochondria replaced cell. Inother embodiments, the cells are obtained by a method comprising: (a)contacting a recipient cell with an agent that reduces mitochondrialfunction; (b) incubating the recipient cell for a sufficient period oftime for the agent to partially reduce the endogenous mitochondrialfunction in the recipient cell; and (c) co-incubating (1) the recipientcell from step (b) in which the endogenous mitochondrial function hasbeen partially reduced, and (2) exogenous mtDNA from a healthy donor,for a sufficient period of time to non-invasively transfer exogenousmtDNA into the recipient cell, thereby generating a mitochondriareplaced cell.

In certain embodiments of the pharmaceutical compositions providedherein, the cells are obtained by a method further comprising furthercomprising contacting the recipient cell with a second active agentprior to co-incubating the recipient cell with exogenous mitochondriaand/or exogenous mtDNA. In some embodiments, the second active agent isselected from the group consisting of large molecules, small molecules,or cell therapies, and the second active agent is optionally selectedfrom the group consisting of rapamycin, NR (Nicotinamide Riboside),bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide(MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thiocticacid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis. In specific embodiments, the activator ofendocytosis is a modulator of cellular metabolism. In other embodiments,the modulator of cellular metabolism comprises nutrient starvation, achemical inhibitor, or a small molecule. In further embodiments, thechemical inhibitor or the small molecule is an mTOR inhibitor. In yetfurther embodiments, said mTOR inhibitor comprises rapamycin or aderivative thereof.

In certain embodiments of the pharmaceutical compositions providedherein, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

In some embodiments of the pharmaceutical compositions provided herein,the cells are T cells. In other embodiments, the cells are hematopoieticstem cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a scheme for generation of a Mitochondria replaced cell(MirC).

FIG. 1B depicts a plasmid construct for the Mitochondrial TargetingSequence (MTS)-XbaI restriction enzyme (XbaIR) plasmid.

FIG. 1C depicts that isolated mitochondrial DNA is digested at multiplesites by the XbaI restriction enzyme, whereas NotI digestion ofmitochondrial DNA resulted in a single fragment, as predicted byCambridge Reference Sequence (CRS) of mitochondrial DNA.

FIG. 1D depicts five XbaIR endonuclease sites (1193, 2953, 7440, 8286,10256) on human mitochondrial DNA as predicted by Cambridge ReferenceSequence (CRS).

FIG. 1E depicts microscopy of human dermal fibroblasts under phasecontrast (left), immunofluorescence of green fluorescence protein(middle), and merged fields (right) after uptake of a fusion MTS-greenfluorescence protein (GFP) plasmid using electroporator (Nucleofector).Top, low magnification. Bottom, high magnification.

FIG. 1F depicts the construct for the pCAGGS-MTS-EGFP-PuroR andpCAGGS-MTS-XbaIR-PuroR plasmids.

FIG. 1G depicts the localization of exogenous transgene productsMTS-EGFP in mitochondria by mitochondria-specific staining withtetramethylrhodamine, methyl ester (TMRM).

FIG. 2A depicts a scheme of the time schedule to compare the MTS-XbaIRendonuclease method (top) with the traditional method using ethidiumbromide (EtBr) (middle), relative to non-contacted cells.

FIG. 2B depicts quantification of human (3-actin (Actb), left columns,and mitochondria DNA (mtDNA), right columns, following contact witheither the MTS-XbaIR endonuclease method or the ethidium bromidetreatment, relative to non-contacted cells. XbaIR resulted in a greaterreduction of mtDNA, compared to EtBr treatment. Actb was used as ahousekeeping gene.

FIG. 2C depicts a greater reduction in mitochondria following exposureto the gene transfer of MTS-XbaIR, relative to EtBr treatment, based onDsRed fluorescence that had been expressed in mitochondria.

FIG. 2D depicts semi-quantification of mitochondrial membrane potentials(surrogate marker for mitochondrial content) in cells contacted with thegene transfer of MTS-XbaIR or EtBr using FACS analyses by using TMRM,and shows that MTS-XbaIR resulted in a greater reduction inmitochondria.

FIG. 2E depicts a time course quantification of transgene expression inthe gene transfer system over fourteen days.

FIG. 2F and FIG. 2G depicts fluorescent images (FIG. 2F) following thetransfer of the plasmid carrying GFP prior to (“pre”) and after (“post”)puromycin section, and quantification of the GFP/Mitochondria ratio(FIG. 2G) demonstrates enrichment of the GFP plasmid post-puromycinselection.

FIG. 3A depicts a scheme of the time schedule for mitochondriareplacement. TF: Gene transfection of XbaIR or Mock; Puro: Puromycin forenrichment of gene transferred cells; U+: Addition of uridine to rescueρ(−) cells devoid of mitochondrial ATP production; Mt Tx: Mitochondriatransfer; NHDF: Normal Human Dermal Fibroblasts, EPC100: Placentalvenous endothelium-derived cell lines.

FIG. 3B depicts reduction in mitochondria on Day 6 following the genetransfer of XbaIR (top), but not following the transfer of the negativecontrol vector expression GFP (bottom), as measured by TMRM staining.

FIG. 3C depicts quantification of mitochondrial DNA copy numbersestimated by qPCR of human 12S rRNA relative to nuclear (3-actin levelsin NHDF cells after gene transfection of XbaIR or GFP transfection.Mitochondria were transferred to recipient cells where indicated (“MtTx”). XbaIR resulted in significant reduction of mitochondrial DNA,which could be rescued to levels equivalent to control treated cellsafter transfer of exogenous mitochondria. N=3, * p<0.01.

FIG. 3D depicts Photographs from time lapse movie: Upper left:Cocultivation of ρ (−) cells with isolated and DsRed-markedmitochondria; Upper right: ρ (−) cells as a control; Lower left:Cocultivation of NHDF with mitochondria; Lower right: Cocultivation ofmock transfectant of NHDF with mitochondria;

FIG. 3E depicts a series of 10 still images from time lapse moviedepicted in FIG. 3D, arranged chronologically, vertically;

FIG. 3F depicts measurement of DsRed labeled mitochondria by FACSanalysis and revealed that the present invention (“DsRed-Mt EPC100”)resulted in increased uptake of exogenous mitochondria compared topreviously described methods.

FIG. 3G and FIG. 311 depict microscopy images of DsRed labeledmitochondria (FIG. 3G) and phase contrast (FIG. 3H) after mitochondriatransfer in ρ(0) cells treated with, or without, antimycin, anddemonstrates that no engulfment of exogenous mitochondria occurred incells with complete destruction of mitochondria;

FIG. 3I depicts a series of 5 still images from time lapse moviedepicted in FIG. 3G, arranged chronologically, vertically.

FIG. 3J depicts quantification of fluorescent intensities ofDsRed-labeled isolated exogenous mitochondria, measured every 24 hoursin ρ (−) cells, or in ρ (−), mock transfected cells, or untreated cells(add on Mt) co-incubated with the Ds-Red mitochondria.

FIG. 4A depicts a scheme for measuring the fate of donor mitochondriafollowing the engulfment by the recipient cells using DsRed markedmitochondria as donors and EGFP marked cells as recipients.

FIG. 4B depicts representative images from movies to observe engulfedexogenous mitochondria (indicated as red) in recipient cells with GFPmarked mitochondria. Movies were recorded by using superfine microscopy,and few fusion images were recognized, and a major of the donormitochondria separately exist to the pre-existing mitochondria.

FIG. 4C depicts three dimensional reconstitutional photograph of thefusion.

FIG. 4D depicts photos of NHDF transferred of gene coding DsRed fusedwith mitochondria transfer signal.

FIG. 4E depicts photos of EPC100 transferred of gene coding EGFP fusedwith TFAM.

FIG. 4F depicts time course of mitochondria transfer using DsRed markedcells as recipients and TFAM targeted EGFP as donor mitochondria.

FIG. 4G depicts exogenous TFAMs were stably engrafted in thepre-existing mitochondria, after the exogenous mitochondria weretransiently contacted with the recipient cell, suggesting thatmitochondrial nucleoids including TFAMs were transferred to thepre-existing mitochondria via the transient contact, analogous tomouth-to-mouth feeding.

FIG. 5A depicts the whole circular mitochondrial DNA with the Cambridgereference sequence (CRS) of human mitochondrial DNA indicatinghypervariable (“HV”) regions 1/2, and 5 primers to identify thedifference between NHDF and EPC100;

FIG. 5B depicts DNA sequencing data for the nucleotides surroundinghmt16362 in NHDF ctrl recipient cells (SEQ ID NO: 1), EPC100 ctrl donorcells (SEQ ID NO: 2), NHDF derived ρ(−) cells without mitochondriareplacement (SEQ ID NO: 3), and NHDF derived ρ(−) cells withmitochondria replacement (SEQ ID NO: 4) and demonstrated that NHDFderived ρ(−) cells with mitochondria replacement (SEQ ID NO: 4) changedfrom A in the original recipient cells to G in the donor mtDNA athmt16362.

FIG. 5C depicts the hmt16318-F (SEQ ID NO: 6) and hmt16414-R (SEQ ID NO:9) set of primers used for amplification of the HV1 region of humanmitochondrial DNA D-Loop (SEQ ID NO: 8) surrounding hmt16362 and theNHDF specific probe (SEQ ID NO: 5) and the EPC100 specific probe (SEQ IDNO: 7) that were designed for the TaqMan SNP genotyping assay.

FIG. 5D depicts quantification of the NHDF specific hmtDNA (left) andEPC100 specific hmtDNA (right) in the parental NHDF and EPC100 celllines, or in NHDF cells treated with XbaIR, with (XbaIR Mt+) or withoutmitochondria (XbaIR Mt−) from EPC100 cells, and revealed that EPC100mitochondria was successfully transferred in XbaIR Mt+ cells, asevaluated by using single nucleotide polymorphism assay (SNP).

FIG. 6A depicts representative oxygraphies from a mitochondriafunctioning assay performed using Oroboros Oxygraph-2k, and demonstratedthat NHDF cells with mitochondria replaced (ρ(−) Mt) (bottom) regainedthe mitochondrial function, relative to control NHDF cells (top), andρ(−) NHDF cells without mitochondrial replacement (middle). The machinedepicts respiratory flow in red line (pmol/sec/1×10⁶ cells, right axis)and oxygen concentrations in blue line (μM, left axis).

FIG. 6B depicts that the respiratory flows (routine, Electron TransferSystem (ETS), ROX), free routine activities (mitochondrial ATPproduction), proton leakage, and coupling efficiency in each stagedemonstrated that mitochondrial replacement in NHDF cells (ρ(−) Mt)regained the mitochondrial function, relative to NHDF control cells, andNHDF without mtDNA replacement (ρ(−)).

FIG. 6C depicts a time-lapse microphotograph, which enabled to estimatecontinuous cell number based on the surface area of cells, anddemonstrated that ρ(−) cells were quiescent state between days 3 to 12,whereas mitochondria replaced cells regained the growth capability afterday 6.

FIG. 6D depicts a scheme of the protocol used to examine the molecularmechanism for macropinocytosis, which involved transfecting NHDF cellswith the MTS-XbaIR-P2A-PuroR plasmid, selecting with puromycin, and thenserum starving the cells for 60 min, or treating the cells with palmiticacid (PA) or rapamycin for 24 hours.

FIG. 6E-FIG. 611 depict quantification of the WES' analysis forphosphorylation of S6 kinase (FIG. 6E) and phosphorylation of AMPK (FIG.6G), and corresponding WES™ blots (FIG. 6F) and (FIG. 6H), respectively,which demonstrated that AMPK is activated and mTOR is completelysuppressed in ρ(−) cells. Rapa: Rapamycin, PA: Palmitic acid, EAA−:Essential amino acid-deficient.

FIG. 6I depicts the protocol used to examine the effect of mTOR mediatedmacropinocytosis in the setting of MirC generation protocol.

FIG. 6J-FIG. 6L depict quantification (FIG. 6J and FIG. 6K) AND FACSanalysis (FIG. 6L) and of DsRed labeled mitochondrial uptake in control(top), mock transfected cells (middle), and ρ(−) cells, with or withoutrapamycin treatment, or with or without palmitic acid (PA) treatment.ρ(−) cells exhibited greater uptake of mitochondria, relative to controlof mock TF cells, and the uptake of mitochondria was significantlyincreased after rapamycin treatment, whereas palmitic acid decreasedmitochondria uptake in ρ(−) cells.

FIG. 7A depicts the whole mtDNA sequence showing the Leigh syndromeassociated mutation of 10158T>C in the respiratory chain complex I (CI)subunit of the ND3 gene in mitochondrial DNA.

FIG. 7B depicts DNA sequencing data for the nucleotides surroundinghmt10158 within ND3 in EPC100 cells (top; SEQ ID NO: 10) and Leighsyndrome (7SP) fibroblasts (bottom; SEQ ID NO: 11), and revealed themutation, 10158T>C, with a mosaic of C in the major wave and T in theminor wave, indicating the heteroplasmy.

FIG. 7C depicts photographs from time lapse movies that demonstratedsimilar behavior in both ρ(−) 7SP fibroblasts, with and withoutexogenous mitochondria, as in NHDF experiments.

FIG. 7D depicts quantification of mitochondrial DNA copy numbersestimated by qPCR of human 12S rRNA relative to nuclear (3-actin levelsin NHDF cells after gene transfection of XbaIR or Mock transfection.Mitochondria were transferred to recipient cells where indicated. XbaIRresulted in significant reduction of mitochondrial DNA, which could berescued by transfer of exogenous mitochondria. (n=3)

FIG. 7E depicts DNA sequencing data for the nucleotides surroundinghmt10158 in 7SP ctrl recipient cells (SEQ ID NO: 14), EPC100 ctrl donorcells (SEQ ID NO: 12), 7SP derived ρ(−) cells without mitochondriareplacement (SEQ ID NO: 13), and 7SP derived ρ(−) cells withmitochondria replacement (SEQ ID NO: 15), and revealed that 7SP ctrlcells are heteroplasmic (majority 10158C; SEQ ID NO: 14), whereas EPC100has only T in the same site in mitochondrial DNA (SEQ ID NO: 12). Theρ(−) cells stem from 7SP cells expressed the same wave as the original(SEQ ID NO: 13), whereas mitochondria replaced 7SP cells demonstrated Tas major wave (SEQ ID NO: 15).

FIG. 7F depicts the hmt10085-F (SEQ ID NO: 17) and hmt10184-R (SEQ IDNO: 20) set of primers used for amplification of ND3 of humanmitochondrial DNA (SEQ ID NO: 16) surrounding the Leigh syndromeassociated SNP at hmt10158, and the EPC100 specific probe (SEQ ID NO:18) and the 7SP specific probe (SEQ ID NO: 19) that were designed forthe TaqMan SNP genotyping assay. The ND3 peptide sequence is alsodepicted (SEQ ID NO: 46).

FIG. 7G depicts quantification of the percentage of hmt10158heteroplasmy in each cell group evaluated by SNP assay, and revealedthat exogenous normal sequence (“healthy”) dominated up to 80% inmitochondria replaced 7SP cells, in spite that the original heteroplasmyof mutant sequence was over 90%. In case of mock transfectant, theheteroplasmy did not significantly change, and maintained the almostsame ratio.

FIG. 7H and FIG. 7I depict quantification of heteroplasmy levelpercentage (FIG. 7H) and absolute mtDNA copy number (FIG. 7I) in threeindependent experiments in 7SP cells treated with mock control andsubjected to mitochondrial transfer.

FIG. 7J depicts a series of 10 still images from the time lapse moviedepicted in FIG. 7C, arranged chronologically, vertically.

FIG. 8A depicts microscopic photos in ρ(−) mitochondria replaced 7SPfibroblasts with time, compared with the original 7SP fibroblasts andρ(−) 7SP fibroblasts, and revealed that the growth of mitochondriareplaced cells recovered to near control level.

FIG. 8B depicts time-lapse-estimated cellular growth in 7SP fibroblasts,ρ(−) 7SP fibroblast, and ρ(−) 7SP fibroblasts with mitochondriareplacement, and revealed that ρ(−) 7SP fibroblasts were quiescent,whereas mitochondria replaced 7SP cells recovered cellular growth tolevels equivalent to the original 7SP fibroblasts around day 12.

FIG. 8C depicts senescence in 7SP fibroblasts around population doublinglevels (PDL) 25, which was extended to about PDL 63 in ρ(−) 7SPfibroblasts with healthy mitochondria replacement performed at PDL 8,indicating the lifespan extension of ρ(−) 7SP fibroblasts with healthymitochondria replacement.

FIG. 8D depicts the increase in PDL produces an increase in cell size(left), which is reverted following mitochondria replacement, and ismaintained even past PDL 50 (right).

FIG. 8E depicts short tandem repeat (STR) assay, which discriminatescells with different origins and identifies contamination of differenttype of cells. The patterns of STR in mitochondria replaced cells indifferent time point were completely identical to that in the original7SP fibroblasts.

FIG. 8F depicts RT-PCR quantification of telomerase in 7SP fibroblastsand mitochondria replaced cells for different PDLs, relative to HeLa andEPC100, indicating that the cells were not transformed into cancercells.

FIG. 9A depicts oxygraphies in 7SP fibroblasts at different PDLsfollowing mitochondria replacement using Oroboros 02k according tocoupling-control protocol (CCP), and the kinetics demonstrated thatmitochondria function dropped at early PDL followed by a gradualrecovery that eventually surpassed the original capability, relative tothe original 7SP fibroblasts as control.

FIG. 9B and FIG. 9C depict that the respiratory flows (routine, ElectronTransfer System (ETS), ROX), free routine activities (mitochondrial ATPproduction), proton leakage, and coupling efficiency (FIG. 9B), as wellas the flux control ratios (FCRs), ROVE, L/E, R/E, and (R-L)/E (FIG. 9C)regained to near control levels in mitochondrial replaced cells (ρ(−)Mt) after approximately PDL30.

FIG. 10A depicts microscopy images of NHDF, 7SP, an 7SP MirC cells underbasal conditions or following reperfusion using H₂O₂, and show that 7SPcells are highly sensitive to H₂O₂ relative to NHDF cells, whereas the7SP MirC are not.

FIG. 10B-FIG. 10D depict FACS analysis (FIG. 10B) and quantification ofAnnexin V (FIG. 10C) and propidium iodine (PI; FIG. 10D) positive cellsfollowing no treatment or treatment with H₂O₂, and demonstrate that 7SPcells are highly sensitive to H₂O₂ relative to NHDF cells, whereas the7SP MirC are not.

FIG. 10E depicts microscopy images of NHDF, 7SP, an 7SP MirC cells underbasal conditions or following starvation conditions (EAA−), and showthat 7SP cells are highly sensitive to starvation conditions relative toNHDF cells whereas the 7SP MirC are not.

FIG. 10F-FIG. 1011 depict FACS analysis (FIG. 10F) and quantification ofAnnexin V (FIG. 10G) and PI (FIG. 10H) positive cells following notreatment or starvation, and demonstrate that 7SP cells are highlysensitive to starvation conditions relative to NHDF cells, whereas the7SP MirC are not.

FIG. 11 depicts quantification of the expression levels ofrepresentative SASP cytokines, IL-6 and IL-8, chemokine, CXCL-1, andgrowth factor, ICAM1 for NHDF, 7SP fibroblast, and 7SPfibroblast-derived MirC cells, whose PDLs were almost the same, about 15to 20, which demonstrated a significant reduction in IL-6, indicating areversal of SASP in the MirC. GAPDH was used for normalization.

FIG. 12A depicts the scheme for generation of induced pluripotent stemcells (iPSCs) from mitochondria replaced 7SP fibroblasts.

FIG. 12B-FIG. 12D depicts alkaline phosphatase (AP) staining andquantification as an indicator of iPSCs, which were generated fromeither 7SP fibroblasts, 7SP fibroblast-derived MirC, or mocktransfectants originated from 7SP fibroblasts. Microscopic (FIG. 12Bleft panel; 7SP fibroblasts, middle panel; 7SP fibroblast-derived MirC,right panel: Mock transfectant of 7SP fibroblast) and macroscopic (FIG.12C left panel; 7SP fibroblasts, middle panel; 7SP fibroblast-derivedMirC, right panel: Mock transfectant of 7SP fibroblast) microscopy of APstained cells, as well as quantification of the AP stained cells (FIG.12D), revealed that mitochondrial replacement in either NHDF or 7SPfibroblasts following XbaIR treatment resulted in increased AP staining.

FIG. 12E depicts colony formation of iPSCs derived from mitochondriareplaced 7SP fibroblasts. Photos of 3 representative colonies in 75 daysand 170 days after gene transfer of reprogramming factors.

FIG. 12F depicts immunohistochemical staining for OCT3/4, NANOG,TRA1-80, and TRA-160 in iPSCs generated from 7SP fibroblasts followingmitochondrial replacement, which are representative markers forpluripotent stem cells;

FIG. 12G depicts mitochondrial DNA copy number in iPSCs derived from 7SPfibroblast derived MirC, compared with the original 7SP fibroblasts andthe standard human iPSCs (201B7) as references, and revealed that iPSCshad limited number of mitochondrial DNA that was similar that of thestandard human iPSCs (201B7).

FIG. 12H and FIG. 12I depict the percentage of heteroplasmy (FIG. 12H)and absolute mtDNA copy number (FIG. 12I) in iPSCs derived from 7SPfibroblast-derived MirC in 170 days after the reprogramming procedure,and revealed that 7SP fibroblast-derived MirC that formed iPSC showednegligible levels of mutated genome sequence, reduced total mtDNA, andnearly 100% donor mtDNA in at least three clones, suggesting that thechange of the heteroplasmy in MirC could be reverted into the originalstate, and different from the mitochondrial replacement therapy in IVF.

FIG. 13A depicts a scheme of the protocol for mitochondrial transferfrom a donor cell to a recipient cell, where the donor cell andrecipient cell are from different stages of a lifespan.

FIG. 13B depicts DNA sequencing data for the nucleotides surroundinghmt16145 in NHDF ctrl recipient cells (SEQ ID NO: 21) which have thegenotype hmt16145 A, and TIG1 ctrl donor cells (SEQ ID NO: 22) whichhave the genotype hmt16145 G.

FIG. 13C depicts quantitation of hmt16145 heteroplasmy level (%) by SNPassay of the cells from mitochondria replaced cells (MirC) (“old” NHDFrecipient cells with mitochondrial transfer of mitochondria from “young”TIG1 donor cells) and indicated that greater than 90% of the mtDNA inthe NHDF derived MirC cells with mitochondria replacement from TIG1derived mitochondria donor cells was hmt16145 G (i.e., from TIG1 mtDNA),whereas 100% of the NHDF ctrl cell's mtDNA was hmt16145 A.

FIG. 13D depicts quantification of the population doubling level (PDL)versus time (days) (left), and doubling time (hours) versus populationdoubling level (right) in recipient NHDF cells transfected with MTS-GFP(“mock”), or MTS-XbaIR (“MirC”) and coincubated with exogenousmitochondria from TIG1 donor cells, or untransfected (“Ctrl”). MirC with“young” donor TIG1 embryonic lung cell (PDL 10) to an “old” normal humandermal fibroblasts (NHDF) recipient cell (PDL 41) showed an extension inlifespan, as indicated by the upward shift in PDL (left) and rightwardshift in PDL (right).

FIG. 13E depicts quantification of the population doubling level (PDL)versus time (days) (left), and doubling time (hours) versus populationdoubling level (right) in normal human dermal fibroblasts transfectedwith MTS-GFP plus mitochondrial transfer (“mock”), MTS-XbaIR plusmitochondrial transfer (“MirC”), or untransfected (“Ctrl”).Mitochondrial transfer from an “old” donor cell (PDL 49) to a “young”recipient cell (PDL<21) showed reduction in lifespan, as indicated bythe downward shift in PDL (left), and leftward shift in PFL (right).

FIG. 14A depicts quality assessment of mRNA generated by in vitrotranscription, as measured by electrophoresis of mRNA for MTS-EGFP andMTS-XbaIR.

FIG. 14B depicts strong expression of the MTS-GFP transgene inmitochondria of T cells 24 hours following electroporation.

FIG. 14C depicts FACS analysis of GFP expression in T cells followingtransfection of the MTS-GFP mRNA by electroporation, and revealed thatGFP expression is present in nearly all T cells.

FIG. 14D depicts FACS analysis of DsRed labeled mitochondria anddemonstrates that the MTS-XbaIR construct robustly degraded theendogenous mitochondria, whereas the MTS-GFP did not.

FIG. 14E depicts a scheme of the protocol design for determining theoptimal time period of mitochondrial co-incubation.

FIG. 14F depicts fluorescent images of control electroporated cells(upper panels) and MTS-GFP electroporated cells (lower panels) at 4 hr,2 days, 4 days, 6 days, and 8 days after electroporation (EP), andindicated the MTS-GFP construct displayed high expression within 4 hourspost-electroporation and was nearly absent by day 6.

FIG. 14G and FIG. 14I1 depict electrophoresis (FIG. 14H) andquantification (FIG. 14G) of GFP in cells receiving the MTS-GFP mRNA,relative to GAPDH. The peak expression occurred at day 4, and expressionwas lost by day 6.

FIG. 14I depicts quantification of XbaIR transcript levels, at 4 hr, day2 (d2), day 4 (d4), day 6 (d6) and day 8 (d8), indicating that thetranscript expressions of the endonuclease were quite highest at 4 hourspost-gene transfer.

FIG. 14J depicts quantification of mitochondrial contents (12S rRNA) incells subjected to MTS-XbaI, and demonstrated that mitochondriadecreased to about 30% by day 2, and was maintained at less than 20%throughout the length of the experiment.

FIG. 15A depicts a scheme of the MirC protocol for human primary Tcells, with electroporation at day 0, analysis at day 2, mitochondria(mt) transfer at day 7, SNP assays at day 9 and 14, and ddPCRheteroplasmy assay at day 14.

FIG. 15B depicts DNA sequencing data for the nucleotides surroundinghmtDNA 218 and hmtDNA 224 of the HV1 region of the human mitochondrialDNA D-loop in human primary NH T cell control recipient cells (top; SEQID NO: 23) and EPC100 control donor cells (bottom; SEQ ID NO: 24).hmtDNA 218 and hmtDNA 224 were C/C (SEQ ID NO: 23) and T/T (SEQ ID NO:24) for T cells and EPC100 cells, respectively.

FIG. 15C depicts the hmtHV1-F (SEQ ID NO: 26) and hmtHV1-R (SEQ ID NO:27) set of primers used for amplification of the HV1 region of humanmitochondrial DNA D-loop (SEQ ID NO: 25) surrounding the SNPs at hmtDNA218 and hmtDNA 224, as well as the SNP assay Primer1-F (SEQ ID NO: 40),the SNP assay-Primer1-R (SEQ ID NO: 41), the N-terminal VIC labeledEPC100 specific probe (SEQ ID NO: 38), and the N-terminal FAM labeled Tcell specific probe (SEQ ID NO: 39) that were designed for the TaqManSNP genotyping assay.

FIG. 15D depicts quantification of the amount of exogenous mtDNA presentin the recipient cells at day 7 and day 12 for mock (MTS-GFP) orMTS-XbaIR (XbaIR) treated cells following coincubation with exogenousmitochondria from donor EPC100 cells. Quantification of recipient anddonor cells was performed as positive controls.

FIG. 15E depicts quantification of respirometry experiments performedusing Oroboros 02k, and demonstrated a recovery of ATP production andcoupling efficiency in human T cell-derived MirC, whereas ρ(−) human Tcells that were generated by XbaIR mRNA transfer with electroporationmaintained the loss of ATP production throughout the experiment.

FIG. 15F and FIG. 15G depict representative raw data usingcoupling-control protocol (CCP), and show that MirC T cells are able torestore mitochondrial respiration.

FIG. 16A depicts comparison viability (left panel) of mouse primary Tcells cultured in RPMI1640 (top) or TexMACS (bottom) at day 2 (left sideof left panel), day 4 (middle of left panel), and day 6 (right side ofleft panel), or CD3 expression (right panel), and demonstrated thatRPMI1640 produced greater viability and higher cell count, as well as aslight increase in CD3 expression, relative to TexMACS culture medium.

FIG. 16B depicts qualitative analysis of GFP expression in T cellsfollowing electroporation (EP) with pmax GFP (middle), or MTS-GFP(right), or without electroporation (left), at 6 hours after EP (topleft panel), day 2 after EP (top right panel), day 4 after EP (bottomleft panel), and day 6 after EP (bottom right panel). Viability was notsignificantly affected following EP with MTS-GFP at day 2 or day 4.

FIG. 16C depicts qPCR quantification of XbaIR levels in T cellselectroporated with the MTS-XbaIR vector at 4 hr, day 2, day 4, and day6 following electroporation and indicated that the XbaIR expressionslowly decreased.

FIG. 16D depicts quantification of 12S rRNA levels in T cellselectroporated with MTS-XbaIR and indicated that the murine mtDNA wasdecreased by approximately 60% by day 4.

FIG. 16E depicts a scheme of the protocol used for MirC generation in Tcells using mitochondrial coincubation on day 5.

FIG. 16F depicts FACS analysis of engulfed DsRed-labeled mitochondria 48hours in the recipient T cells, following the co-incubation withisolated DsRed-labeled mitochondria and revealed a significant positivefraction (9.73%) of T cells expressing exogenous mitochondria inMTS-XbaIR (right), compared with 0.43% in control cells withoutelectroporation (i.e., “add-on”).

FIG. 17A depicts DNA sequencing data for the nucleotides surrounding ND1in mouse mtDNA C57BL6 recipient cells (“BL6”; top; SEQ ID NO: 34) whichhave the genotype mmt2766-A and mmt2767-T, and NZB donor cells (bottom;SEQ ID NO: 35), which have the genotype mmt2766-G and mmt2767-C.

FIG. 17B depicts the 2716-F (SEQ ID NO: 28) and 2883-R (SEQ ID NO: 33)set of primers used for amplification of ND1 of mouse mitochondrial DNA(SEQ ID NO: 32) surrounding the polymorphic nucleotides mmt2766 andmmt2767, and the BL6 specific probe (SEQ ID NO: 29) and the NZB specificprobe (SEQ ID NO: 31), that were designed for the TaqMan SNP genotypingassay, as well as the BamH1-mND1-F primer (SEQ ID NO: 30) used to clonethe nucleotide sequence in a plasmid for generation of a standard curveto enable absolute quantification. The ND1 peptide sequence is alsodepicted (SEQ ID NO: 47).

FIG. 17C depicts quantification of mouse mtND1 heteroplasmy levels inBL6 recipient cells at day 7 and day 12 following controlelectroporation (columns 1 and 2, respectively) or MTS-XbaIelectroporation and coincubation with isolated mitochondria from NZBcells (columns 3 and 4, respectively). Basal levels of BL6 (column 5)and NZB (column 6) cells were measured as controls.

FIG. 17D depicts measurement of telomere length following the treatmentof old murine cells with the MTS-XbaIR mRNA and co-incubation withexogenous mitochondria from the young donor cells to generate the MirC(Young to Old: YtoO) and revealed an increase in the length of telomeresin MirC compared to the parental “Old” cells.

FIG. 17E depicts measurement of SASP associated cytokines CXCL1, ICAM1,IL-6, and IL-8 in the parental old T cell, or the MirC-derived T cell,and indicated that CXCL1 and IL6 were lower in the MirC-derived T cells.

FIG. 17F depicts measurement of DNA damage response in the MirC and theoriginal T cells using the histone 2 A (H2A) phosphorylation antibody,which indicated that the positive fraction for DDR was lower in the MirC(1.53%), compared with the original T cells (4.75%).

FIG. 18A depicts a scheme of the in vivo ACT experiment using old micewith ACT of T cells from young mouse (Group 1), old mice with ACT (Group2) or old mice with ACT of MirC derived from a T cell of an old mousetransferred with exogenous mitochondria from a young mouse (Group 3).

FIG. 18B depicts a representative image of tumor growth imagingperformed during the experimental protocol.

FIG. 18C depicts the body weight of the mock, young T cell, or MirCgroups, and reveals that no significant difference between the threegroups was observed during the 25 days experiment.

FIG. 18D and FIG. 18E depict quantification of the individual (FIG. 18D)and mean (FIG. 18E) cancer mass size, and revealed that the MirC groupreduced cancer mass size to levels equivalent to the Young T cell group(lower lines), whereas the mock group increased in cancer massthroughout the length of the experiment (top lines).

FIG. 18F depicts a scheme of the protocol used to analyze the present ofinfused T cells in the animals.

FIG. 18G depicts FACS analysis of peripheral blood (left panels) orspleen (right panels). Negative controls using C57BL/6 mice (left upperpanel), and positive controls using GFP transgenic mice (left lowerpanel) were generated for both the peripheral blood and the spleen.Positive fractions of T cells expressing GFP fluorescence wererecognized in both the peripheral blood and spleen, 0.057% and 0.9%,respectively.

FIG. 18H depicts immunofluorescence images of the transferred T cellsdetected in the mice on day 6 following transplantation.

FIG. 18I depicts the percentage of chimerism following infusion of theexogenous T cells in peripheral blood (PB) or the spleen after injectionof 1×10⁷ or 2×10⁷ cells.

FIG. 19A and FIG. 19B depict evaluation of MTS-GFP transfection intohematopoietic cells (HSCs) using the X-001, Y-001, and T-030 programs(MTS-GFP1, 2, and 3, respectively) or pmax GFP as a positive control orCtl EP as a negative control by microscopy (FIG. 19A) or FACS (FIG.19B), and show that MTS-GFP1 was the optimal protocol forelectroporating HSCs.

FIG. 19C depicts 3-D confocal fluorescent imaging of the bonemarrow-derived Sca-1 cells 48 hours after the co-incubation withDsRed-labeled mitochondria from EPC100 cells, and showed that theexogenous mitochondria were engulfed.

FIG. 19D depicts quantification of the mitochondrial transfer efficiencyby FACS analysis of DsRed fluorescence, and revealed that asubpopulation of about 10% of the Sca-1 exhibited a right ward shift ofthe fluorescent.

FIG. 19E depicts the scheme used to generate HSC derived MirC bycoincubating with exogenous mitochondria on day 4 and analyzing the MirCon day 6 by SNP assay.

FIG. 19F depicts the FACS sorting for the c-kit+, Sca-1+, Lineage−,CD34− (called as KSLC) fraction of cells.

FIG. 19G depicts that the doubling time of the KSLC fraction was 19hours.

FIG. 19I depicts the scheme used to evaluate HSC derived MirC.

FIG. 19I depicts quantification of the murine mtND1 heteroplasmy levelpercentage in murine KSLC-derived MirC or the parental recipient BL6cells or NZB donor cells, and demonstrated that the MirC derived HSCexpressed 99.9% of the polymorphism genotype of the donor cells on day 6following the MTS-XbaI mRNA transfer with electroporation.

FIG. 20A depicts a 2-D plot of droplet digital PCR results in whichsequences for mutated mtDNA and non-mutated mtDNA were analyzed innormal human dermal fibroblasts for tRNA Leu 3243 A>G, and show onlydetection of the non-mutant sequence (lower right quadrant) and nodetection of the mutant sequence (upper left quadrant).

FIG. 20B depicts a 2-D plot of droplet digital PCR results in whichsequences for mutated mtDNA and non-mutated mtDNA were analyzed innormal human dermal fibroblasts for ND3 10158 T>C, and show onlydetection of the non-mutant sequence (lower right quadrant) and nodetection of the mutant sequence (upper left quadrant).

FIG. 20C depicts a 2-D plot of droplet digital PCR results in whichsequences for mutated mtDNA and non-mutated mtDNA were analyzed innormal human dermal fibroblasts for ATP6 9185 T>C, and show onlydetection of the non-mutant sequence (lower right quadrant) and nodetection of the mutant sequence (upper left quadrant).

FIG. 20D depicts a 2-D plot of droplet digital PCR results in whichsequences for mutated mtDNA and non-mutated mtDNA were analyzed inprimary skin fibroblasts from a patient with MELAS having an mtDNAA3243G mutation, and show the majority of cells had homoplasmy ofmutated mtDNA (upper left quadrant).

FIG. 20E depicts a 2-D plot of droplet digital PCR results in whichsequences for mutated mtDNA and non-mutated mtDNA were analyzed inprimary skin fibroblasts from a patient with Leigh Syndrome having amtDNA T10158C mutation of Complex I, ND3 gene, and showed a minorportion of double positive cells with heteroplasmy in a single celllevel (upper right quadrant), a major population of homoplasmy ofmutated mtDNA (lower right), and no population with homoplasmy ofnon-mutated mtDNA (upper left).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are novel and enhanced methods of generating amitochondria replaced cell (MirC) that do not require complete removalof endogenous mtDNA, and can optionally be performed using reagents thatare compatible with clinical use. In addition, in certain embodimentsprovided herein are methods of treatment involving administering atherapeutically effective amount of the MirC generated using the methodsprovided herein.

Also provided are compositions that include one or more mitochondriareplaced cells obtained by the methods provided herein. In certainembodiments, the compositions can also include a second active agentthat enhances the uptake of exogenous mitochondria, exogenous mtDNA, ora combination thereof, and/or an agent that reduces endogenous mtDNAcopy number or reduces endogenous mitochondrial function. In furtherembodiments, the compositions can also include exogenous mitochondriaand/or exogenous mtDNA, one or more recipient cells, or a combinationthereof. In one specific embodiment, provided herein are methods andcompositions for use in the treatment of a disease or disorderassociated with dysfunctional mitochondria. However, it is understoodthat the methods and compositions provided herein can also be used todelay senescence, extend the lifespan, or enhance the function of a cellthat has functional mitochondria, and is not limited to replacement ofdysfunctional mitochondria. Furthermore, the methods and compositionsprovided herein can also be used to replace functional mitochondria withexogenous mitochondria that is dysfunctional or exhausted, for example,to generate a disease model.

5.1 Definitions

Unless particularly defined otherwise, all terms including technical andscientific terms used in this application have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In general, the nomenclatures used in thisspecification and the experimental methods described below are widelyknown and generally used in the related art.

As used herein, the term “mitochondria replaced cell” or MirC isintended to mean a cell having the substitution of endogenousmitochondria and/or mtDNA with exogenous mitochondria and/or mtDNA. Forexample, an exemplary mitochondria replaced cell (MirC) involves thesubstitution of endogenous mtDNA that encodes dysfunctionalmitochondria, such as mtDNA originating from a subject having amitochondrial disease or disorder, with exogenous mtDNA that encodesfunctional mitochondria, such as mtDNA originating from a healthysubject. Exemplary MirC can also include a cell with endogenousmitochondria substituted with exogenous mitochondria. However, it isunderstood that the substitution of the endogenous mitochondria and/ormtDNA can also include, for example, functional endogenous mtDNA fromone cell, such as from an old cell, that is substituted with functionalexogenous mtDNA from a different cell, such as from a healthier cellthat is from a young subject. It is further understood that healthyendogenous mitochondria and/or mtDNA can also be substituted withdysfunctional exogenous mitochondria and/or exogenous mtDNA such as, forexample, to mimic a mitochondrial disease or disorder. Replacement neednot result in a complete substitution of all the endogenous mitochondriain a cell, and that exemplary mitochondria and/or mtDNA replacementinvolves substitution of about 5% of more, about 10% or more, about 20%or more, about 30% or more, about 40% or more, about 50% or more, about60% or more, about 70% or more, about 80% or more, about 90% or more, orabout 95% or more of the endogenous mitochondria and/or mtDNA.

As used herein, the term “recipient cell,” “acceptor cell,” and “hostcell” are interchangeable and refer to a cell receiving the exogenousmitochondria and/or mtDNA. In some embodiments, the exogenousmitochondria and/or mtDNA is from isolated mitochondria from a donorcell. In some embodiments, the donor cells and the recipient cells maybe different or identical. In some embodiments, the donor cells and therecipient cells come from different or the same species. In someembodiments, the donor cells and the recipient cells come from differentor the same tissues.

As used herein, the term “healthy donor” is intended to mean a donorthat does not have a mitochondrial disease or disorder, age-relateddisease, or otherwise dysfunctional mitochondria. In preferredembodiments, a healthy donor has a wild-type mtDNA sequence, relative tothe Cambridge Reference Sequence of the mitochondrial genome.

As used herein, the terms “treat,” “treating,” and “treatment” refer toreduction in severity, progression, spread, and/or frequency ofsymptoms, elimination of symptoms and/or underlying cause, prevention ofthe occurrence of symptoms and/or their underlying cause, andimprovement or remediation of damage. “Treatment” is meant to includetherapeutic treatment as well as prophylactic, or suppressive measuresfor the condition, disease or disorder.

As used herein, the term “agent” when used in reference to depletingreducing mtDNA refers to an enzyme or compound that is capable ofreducing mtDNA. Preferred agents include restriction enzymes, such asXbaI, that cleave mtDNA at one or more sites, without producing toxicityin the recipient cell. However, agents can also include an enzyme orcompound that inhibit mtDNA synthesis or selectively promote degradationof the mitochondria.

As used herein, the terms “reduce,” or “decrease” generally means adecrease of at least 5%, for example a decrease by at least about 10%,or at least about 20%, or at least about 30%, or at least about 40%, orat least about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90%, or any decrease between 5%-99%as compared to a reference level, as that term is defined herein. It isunderstood that a partial reduction or an agent that partially reducesendogenous mtDNA or decrease, as used herein, does not result in acomplete depletion of all endogenous mtDNA (i.e., ρ0 cells). The term“increase” as used herein generally means an increase of at least 5%,for example an increase by at least about 10%, or at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90%, or more than 90%.

As used herein, the term “endogenous” refers to originating or derivedinternally. For example, endogenous mitochondria are mitochondria thatare native to a cell.

As used herein, the term “exogenous” refers to cellular material (e.g.,mitochondria or mtDNA) that is non-native to the host, such as cellularmaterial that is derived externally. “Externally” typically means from adifferent source. For example, mitochondrial genomes are exogenous tohost cells or host mitochondria when the mitochondrial genomes originatefrom different cell types or different species than the host cells orhost mitochondria. In addition, “exogenous” can also refer tomitochondrial genomes that are removed from mitochondria, manipulated,and returned to the same mitochondria.

As used herein, the term “sufficient period of time” refers to an amountof time that produces the desired results. It is understood that thesufficient period of time will vary according to the experimentalconditions, including but not limited to, the temperature, the amount ofreagent used, and the cell type. Exemplary protocols are providedthroughout as guidelines for the “sufficient period of time,” and aperson skilled in the art would be able to identify the period of timethat is sufficient without undue experimentation.

As used herein, the term “majority” is intended to mean the greatestamount, relative to the other amounts being compared. An exemplarymajority when comparing two groups, is an amount that is any integergreater than about 50% or more, about 60% or more, about 70% or more,about 80% or more, or about 90% or more, or about 95% or more, of thetotal population, including any integer in-between. It is understoodthat the majority will depend on the total population being compared,and can be amounts lower than 50% when there are three or more groupsbeing compared.

As used herein, the term “non-invasively” when used in reference to thetransfer of exogenous material is intended to mean without the use ofinvasive instruments (e.g., nanoblade or electroporation), physicalforce (e.g., centrifugation), or harmful culture conditions (e.g.,thermal shock). In preferred embodiments, the non-invasive transferprocedure involves co-incubation of a recipient cell and donormitochondria.

As used herein, the term “subject in need of mitochondrial replacement,”is intended to mean a subject that has or is predisposed to having adysfunctional mitochondria. The subject in need of mitochondrialreplacement may be asymptomatic and in need of preventative care. Thesubject in need of mitochondrial replacement may also be symptomatic andin need of treatment. In certain embodiments, the subject in need ofmitochondrial replacement has dysfunctional mitochondria that is not theresult of an age-related disease or a mitochondrial disease or disorder.

As used herein, the term “subject” is intended to mean a mammal. Asubject can be a human or a non-human mammal, such as a dog, cat, bovid,equine, mouse, rat, rabbit, or transgenic species thereof. It isunderstood that a “subject” can also refer to a “patient,” such as ahuman patient.

As used herein, the term “effective amount” refers to the amount of acomposition of the invention effective to modulate, treat, or ameliorateany disease or disorder associated with heteroplasmy and/ordysfunctional mitochondria. As such, an effective amount can include,for example, a therapeutically effective amount, which refers to aneffective amount in a therapy, or a biologically effective amount, whichrefers to an effective amount for a biological effect. The terms“therapeutically effective amount” and “effective amount” can encompassan amount that improves overall therapy, reduces or avoids symptoms orcauses of disease or disorder, or enhances the therapeutic efficacy ofanother therapeutic agent. The amount of a given composition that willcorrespond to such an amount will vary depending upon various factors,such as the given composition, the pharmaceutical formulation, the routeof administration, the type of condition, disease or disorder, theidentity of the subject or host being treated, and the like, but cannevertheless be routinely determined by one skilled in the art. Asdefined herein, a therapeutically effective amount of an agent may bereadily determined by one of ordinary skill by routine methods known inthe art.

As used herein, the term “age-related disease” refers to any number ofconditions attributable to advancement in age. These conditions include,without limitation, osteoporosis, bone loss, arthritis, stiffeningjoints, cataracts, macular degeneration, metabolic diseases includingdiabetes mellitus, neurodegenerative diseases including Alzheimer'sDisease and Parkinson's Disease, immunosenescence, and heart diseaseincluding atherosclerosis and dyslipidemia. The phrase “age relateddisease” further encompasses neurodegenerative diseases, such asAlzheimer's Disease and related disorders, ALS, Huntington's disease,Parkinson's Disease, and cancer.

As used herein, the term “autoimmune disease” is intended to mean adisease or disorder arising from immune reactions directed against anindividual's own tissues, organs or a manifestation thereof or aresulting condition therefrom. An autoimmune disease can refer to acondition that results from, or is aggravated by, the production ofautoantibodies that are reactive with an autoimmune antigen or epitopethereof. An autoimmune disease can be tissue- or organ-specific, or itcan be a systemic autoimmune disease. Systemic autoimmune diseasesinclude connective tissue diseases (CTD), such as systemic lupuserythematosus (lupus; SLE), mixed connective tissue disease systemicsclerosis, polymyositis (PM), dermatomyositis (DM), and Sjögren'ssyndrome (SS). Additional exemplary autoimmune diseases further includerheumatoid arthritis, and anti-neutrophil cytoplasmic antibody (ANCA)polyangiitis.

As used herein, the term “genetic disease” refers to a disease caused byan abnormality, such as a mutation, in the nuclear genome. Exemplarygenetic diseases include, but are not limited to, Hutchinson-GilfordProgeria Syndrome, Werner Syndrome, and Huntington's disease.

As used herein, the term “cancer” includes but is not limited to, solidcancer and blood borne cancer. The terms “cancer” and “cancerous” referto or describe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth.

As used herein, the terms “mitochondrial disease or disorder” and“mitochondrial disorder” are interchangeable and refer to a group ofconditions caused by inherited or acquired damage to the mitochondriacausing an energy shortage within those areas of the body. Exemplaryorgans effected by mitochondrial disease or disorder include those thatconsume large amounts of energy such as the liver, muscles, brain, eye,ear, and the heart. The result is often liver failure, muscle weakness,fatigue, and problems with the heart, eyes, and various other systems.

As used herein, the term “mitochondrial DNA abnormalities” refer tomutations in mitochondrial genes whose products localize to themitochondrion, and not observed in the cells of healthy subjects.Exemplary diseases associated with mitochondrial DNA abnormalitiesinclude, for example, chronic progressive external ophthalmoplegia(CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes anddeafness (DAD), leber hereditary optic neuropathy (LHON), LHON-plus,neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP),maternally-inherited Leigh syndrome (MILS) also known as Leigh syndromecaused by mutant mtDNA, mitochondrial encephalomyopathy, lacticacidosis, and stroke-like episodes (MELAS), myoclonic epilepsy andragged-red fiber disease (MERRF), familial bilateral striatalnecrosis/striatonigral degeneration (FBSN), Luft disease,aminoglycoside-induced Deafness (AID), and multiple deletions ofmitochondrial DNA syndrome.

As used herein, the term “nuclear DNA abnormalities” within the contextof mitochondrial disease or disorder refer to mutations or changes inthe coding sequence of nuclear genes whose products localize to themitochondrion. Exemplary mitochondrial disease or disorders associatedwith nuclear mutations include Mitochondrial DNA depletion syndrome-4A,mitochondrial recessive ataxia syndrome (MIRAS), mitochondrialneurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNAdepletion syndrome (MTDPS), DNA polymerase gamma (POLG)-relateddisorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO),leukoencephalopathy with brainstem and spinal cord involvement andlactate elevation (LBSL), co-enzyme Q10 deficiency, Leigh syndrome(caused by nuclear mutations), mitochondrial complex abnormalities,fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC)deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenasecomplex deficiency (PDHC), pyruvate carboxylase deficiency (PCD),carnitine palmitoyltransferase I (CPT I) deficiency, carnitinepalmitoyltransferase II (CPT II) deficiency, carnitine-acyl-carnitine(CACT) deficiency, autosomal dominant-/autosomal recessive-progressiveexternal ophthalmoplegia (ad-/ar-PEO), infantile onset spinal cerebellaratrophy (IOSCA), mitochondrial myopathy (MM) spinal muscular atrophy(SMA), growth retardation, aminoaciduria, cholestasis, iron overload,early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

As used herein, the term “dysfunctional mitochondria” refer tomitochondria that are in opposition to functional mitochondria.Exemplary dysfunctional mitochondria include mitochondria that areincapable of synthesizing or synthesize insufficient amounts of ATP byoxidative phosphorylation. As used herein, the term “functionalmitochondria” refers to mitochondria that consume oxygen and produceATP.

As used herein, the term “mutation” refers to any changing of thestructure of a gene, resulting in a variant (also called “mutant”) form.Mutations in a gene may be caused by the alternation of single base inDNA, or the deletion, insertion, or rearrangement of larger sections ofgenes or chromosomes. In some embodiments, the mutation can affect thefunction or the resulting protein. For example, a mutation in a singlenucleotide of DNA (i.e., point mutation) in the coding region of aprotein can result in a codon that encodes for a different amino acid(i.e., missense mutation). It is understood that this different aminoacid can alter the structure of the protein, and that in certaincircumstances, as described herein, can alter the function of theorganelle, such as the mitochondrion.

As used herein, the terms “heteroplasmy” and “heteroplasmic” refer tothe occurrence of more than one type of mitochondrial DNA genome in anindividual or sample. Varying degrees of heteroplasmy are associatedwith varying degrees of the physiological conditions described herein.Heteroplasmy may be identified by means known to the art, and theseverity of the physiological condition associated with specificnucleotide alleles is expected to vary with the percentage of suchassociated alleles within the individual.

As used herein, the term “wild-type” when used in the context ofmitochondrial DNA refers to the genotype of the typical form of aspecies as it occurs in nature. An exemplary reference genome for thewild-type human mtDNA genome includes the Cambridge Reference Sequence(CRS).

As used herein, the term “old” or “older” is intended to mean that thesource of the mtDNA is from a subject that is greater in age than therecipient cell, or from a cell in a population of cells that havedoubled their population a greater number of times since their culturein vitro (i.e., population doubling level, PDL) relative to therecipient cell.

As used herein, the term “young” or “younger” is intended to mean thatthe source of the mtDNA is from a subject that is lower in age than therecipient cell, or from a cell in a population of cells that havedoubled their population a fewer number of times since their culture invitro (i.e., population doubling level, PDL) relative to the recipientcell.

As used herein, the term “isolated” when used in reference tomitochondria refers to mitochondria that have been physically separatedor removed from the other cellular components of its natural biologicalenvironment.

As used herein, the terms “intact” and “intact mitochondria” refers tomitochondria comprising an outer and an inner membrane, aninter-membrane space, the cristae (formed by the inner membrane) and thematrix. Exemplary intact mitochondria contain mtDNA. In preferredembodiments, intact mitochondria are functional mitochondria. However,it is understood that intact dysfunctional mitochondria can also be usedin the present invention.

As used herein, the term “autologous” is intended to mean biologicalcompositions obtained from the same subject.

As used herein, the term “allogeneic” is intended to mean biologicalcompositions obtained from the same species, but a different genotypethan that of the subject receiving the biological composition.

As used herein, the term “animal cell” is intended to mean any cell froma eukaryotic organism. It is understood that an animal cell can includemammalian and non-mammalian species, such as amphibians, fish, insects(e.g., Drosophila), and worms (e.g., Caenorhabditis elegans).

As used herein, the term “fusion protein” refers to a sequence of aminoacids, predominantly, but not necessarily, connected to each other bypeptidic bonds, wherein a part of the sequence is derived (i.e., hassequence similarity to sequences) from one origin (native or synthetic)and another part of the sequence is derived from one or more otherorigin. Exemplary fusion proteins can be prepared by construction of anexpression vector that codes for the whole of the fusion protein (codingfor both sections, such as a mitochondrial-targeted sequence and anendonuclease) so that essentially all the bonds are peptidic bonds. Itis also understood that the fusion may be made by chemical conjugation,such as by using any of the known methodologies used for conjugatingpeptides.

As used herein, the terms “mitochondrial-targeted sequence (MTS)” and“mitochondrial targeting sequence (MTS)” are interchangeable and referto any amino acid sequence capable of causing the transport of anenzyme, peptide, sequence, or compound attached to it into themitochondria. In certain embodiments, the MTS is a human MTS. In anotherembodiment, the MTS is from another species. Non-limiting examples ofsuch sequences are the cytochrome c oxidase subunit X (COX10) MTS(MAASPHTLSSRLLTGCVGGSVWYLERRT, SEQ ID NO: 36), and the cytochrome coxidase subunit VIII (COX8) MTS (MSVLTPLLLRSLTGSARRLMVPRA, SEQ ID NO:37). Additional non-limiting examples of MTS sequences are the naturalMTS of each individual mitochondrial protein that is encoded by thenuclear DNA, translated (produced) in the cytoplasm and transported intothe mitochondria, as well as citrate synthase (cs), lipoamidedeydrogenase (LAD), and C6ORF66 (ORF). The various MTS may beexchangeable for each mitochondrial enzyme among themselves. Eachpossibility represents a separate embodiment of the fusion protein foruse of the present invention.

As used herein, the term “small molecule” refers to a compound thataffects a biological process and has molecular weight of about 900Daltons or lower. An exemplary small molecule had a molecular weightbetween about 300 and about 700 daltons.

As used herein, the terms “about” or “approximately” when used inconjunction with a number refer to any number within 1, 5, 10, 15 or 20%of the referenced number.

As used herein, the term “somatic cell” refers to any differentiatedcell forming the body of an organism, apart from stem cells, progenitorcells, and germline cells (i.e., ovogonies and spermatogonies) and thecells derived therefrom (e.g., oocyte, spermatozoa). For instance,internal organs, skin, bones, blood, and connective tissue are all madeup of somatic cells. Somatic cells are obtained from animals, preferablyhuman subjects, and cultured according to standard cell cultureprotocols available to those of ordinary skill in the art.

As used herein, the term “endocytosis pathway” refers to the cellularprocess in which cells take in molecules from their surroundings. Theendocytosis pathway can be “clathrin-dependent,” which requires therecruitment of clathrin to help curve the plasma membrane into thevesicle which absorbs the molecules, or “clathrin-independent,” whichdoes not require the recruitment of clathrin. An exemplary type ofclathrin-independent endocytosis includes, for example,macropinocytosis. The term “activator of endocytosis,” as used herein,refers to agents that, e.g., induce or activate the endocytosis pathway,or process, such that the endocytosis pathway is increased. An exemplary“activator of endocytosis” increases mitochondrial uptake from theextracellular environment.

As used herein, the term “macropinocytosis” refers to aclathrin-independent form of endocytosis that mediates the non-selectiveuptake of solute molecules, nutrients and antigens.

As used herein, the term “compound” refers to a compound capable ofeffecting a desired biological function. The term includes, but is notlimited to, DNA, RNA, protein, polypeptides, and other compoundsincluding growth factors, cytokines, hormones or small molecules.

As used herein, the term “peptide” the terms “peptide,” “polypeptide”and “protein” are used interchangeably and in their broadest sense torefer to constrained (that is, having some element of structure as, forexample, the presence of amino acids which initiate a β turn or βpleated sheet, or for example, cyclized by the presence of disulfidebonded Cys residues) or unconstrained (e.g., linear or unstructured)amino acid sequences. The amino acids making up the polypeptide may benaturally derived, or may be synthetic. The polypeptide can be purifiedfrom a biological sample. The polypeptide, protein, or peptide alsoencompasses modified polypeptides, proteins, and peptides, e.g.,glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides,lipoproteins, or lipopeptides.

As used herein, the terms “modulate,” “modulation,” “modulator,” and“modulating” are intended to mean a change in the character orcomposition of the basal, homeostatic state.

An exemplary modulation includes altering cellular metabolism bydisrupting the homeostasis, such that cellular metabolism issignificantly reduced. The term “modulator” includes inhibitors andactivators. Inhibitors are agents that, e.g., inhibit expression ormodification of a desired protein, pathway, or process, or bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity ofthe described target protein, pathway, or process. In certainembodiments, inhibitors are antagonists of the target protein, pathway,or process. Activators are agents that, e.g., induce or activate theexpression or modification of a described target protein, pathway, orprocess, or bind to, stimulate, increase, open, activate, facilitate,enhance activation of inhibitor activity, sensitize or up regulate theactivity of described target protein (or encoding polynucleotide),pathway, or process. In certain embodiments, an activator is an agonistof the target protein, pathway, or process. Modulators include naturallyoccurring and synthetic ligands, antagonists and agonists (e.g., smallchemical molecules, antibodies and the like that function as eitheragonists or antagonists). It is further understood that modulators canbe biological (e.g., antibodies), or chemical.

As used herein, the term “prior to” is intended to mean a period of timepreceding the initiation of an event, such that it is a sufficientlength of time to achieve and sustain a desired result (e.g., antibioticselection) or effect (e.g., biological effect) without the desiredresult or effect completely dissipating before the intended event isinitiated. For instance, in an exemplary situation, it is understoodthat modulating cellular metabolism prior to transfer of an exogenousmitochondria and/or exogenous mtDNA would involve a sufficient period oftime to, for example, exhibit a desired biological effect (e.g.,increase phosphorylation of S6 kinase), without the biological effectreverting back to the homeostatic state before the transfer of exogenousmitochondria and/or exogenous mtDNA occurs.

As used herein, the term “nutrient stress” refers to nutrient deficiencyor nutrient starvation conditions sufficient to produce perturbations inthe cellular homeostasis, such as induction of autophagy, AMPKsignaling, and/or mTOR signaling pathways. Exemplary nutrient stressconditions include serum starvation, removal of essential amino acids,and/or disruption of metabolic pathways.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, or compounds producedsynthetically, which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. As used herein in the context of a polynucleotidesequence, the term “bases” (or “base”) is synonymous with “nucleotides”(or “nucleotide”), i.e., the monomer subunit of a polynucleotide. Theabbreviation “A,” when used in reference to a nucleotide is intended tomean adenine (A). The abbreviation “G,” when used in reference to anucleotide is intended to mean Guanine (G). The abbreviation “C,” whenused in reference to a nucleotide is intended to mean Cytosine (C). Theabbreviation “T,” when used in reference to a nucleotide is intended tomean Thymine (T).

The term “pharmaceutically acceptable” when used in reference to acarrier, is intended to mean that the carrier, diluent or excipient mustbe compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The practice of the embodiments provided herein will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, and immunology, which are within the skill of thoseworking in the art. Such techniques are explained fully in theliterature. Examples of particularly suitable texts for consultationinclude the following: Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Ed., Cold Spring Harbor Laboratory, New York (2001);Ausubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1999); Glover, ed., DNA Cloning, Volumes I and II(1985); Gait, ed., Oligonucleotide Synthesis (1984); Hames & Higgins,eds., Nucleic Acid Hybridization (1984); Hames & Higgins, eds.,Transcription and Translation (1984); Freshney, ed., Animal CellCulture: Immobilized Cells and Enzymes (IRL Press, 1986); Kallen et al,Plant Molecular Biology—A Laboratory Manual (Ed. by Melody S. Clark;Springer-Verlag, 1997); Immunochemical Methods in Cell and MolecularBiology (Academic Press, London); Scopes, Protein Purification:Principles and Practice (Springer Verlag, N.Y., 2d ed. 1987); and Weir &Blackwell, eds., Handbook of Experimental Immunology, Volumes I-IV(1986).

5.2 Methods of Generating a Mitochondria Replaced Cell (MirC)

The present invention is based, in part, on the discovery that any agentthat reduces the function of endogenous mitochondria, including an agentthat reduces endogenous mitochondrial DNA (mtDNA), may enhance thenon-invasive transfer of exogenous mitochondria. However, the completedepletion of the endogenous mtDNA, such as with ρ(0) cells, preventsthis enhancement. This is because the non-invasive transfer of exogenousmitochondria is energy dependent, and a complete depletion of theendogenous mtDNA greatly limits the energy available to facilitate thenon-invasive transfer process. Similarly, the non-invasive transfer ofexogenous mitochondria is also inefficient when the mitochondriafunction and/or mtDNA is unperturbed, for example, when mitochondria ismerely co-incubated (i.e., “add-on”) or added by centrifugation.

Thus, provided herein are methods of generating a mitochondria replacedcell (MirC), that can include (a) contacting a recipient cell with anagent that reduces endogenous mtDNA copy number or an agent that reducesmitochondrial function; (b) incubating the recipient cell for asufficient period of time for the agent to partially reduce theendogenous mtDNA copy number or partially reduce the endogenousmitochondrial function in the recipient cell, respectively; and (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA, or the endogenous mitochondrial function,respectively, has been partially reduced, and (2) exogenous mitochondriafrom a healthy donor, for a sufficient period of time to non-invasivelytransfer exogenous mitochondria into the recipient cell, therebygenerating a mitochondria replaced cell. Also provided herein, is amethod of generating a mitochondria replaced cell, that includesperforming steps (a) and (b) as described above, and then (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA or the endogenous mitochondrial function, respectively,has been partially reduced, and (2) exogenous mtDNA from a healthydonor, for a sufficient period of time to non-invasively transferexogenous mtDNA into the recipient cell, thereby generating amitochondria replaced cell. In certain embodiments, the exogenous mtDNAis transferred via exogenous mitochondria.

The generation of a MirC can be a useful strategy for a variety ofapplications. By way of example, the transfer of exogenous mitochondria,exogenous mtDNA, or a combination thereof into a recipient cell can beuseful in, for example, replacing endogenous mitochondria that isdysfunctional and/or comprised of mutant mtDNA with functionalmitochondria, such as mitochondria comprised of wild-type mtDNA. Incertain embodiments, the methods provided herein are performed in arecipient cell that has endogenous mtDNA that encodes for dysfunctionalmitochondria. In specific embodiments, the endogenous mtDNA is mutantmtDNA. In certain embodiments, the endogenous mtDNA is heteroplasmic andcomprised of both wild-type mtDNA and mutant mtDNA.

As described above, in certain applications, the transfer of exogenousmitochondria, exogenous mtDNA, or a combination thereof can involve thetransfer of functional mitochondria or wild-type mtDNA to replaceendogenous mitochondria that is, for example, dysfunctional or comprisedof mutant mtDNA. Accordingly, in certain embodiments, the exogenousmtDNA is wild-type mtDNA. In other embodiments, the endogenousmitochondria of the recipient cell has wild-type mtDNA, anddysfunctional endogenous mitochondria. For example, exemplarydysfunctional mitochondria of the recipient cell with wild-type mtDNAcan include mutant nuclear DNA that encode for mitochondrial proteins,or dysfunctional mitochondria that arises due to a secondary effect,such as aging or disease.

The endogenous mitochondria that is dysfunctional, comprised of mutantmtDNA, or a combination thereof can therefore be replaced using themethods described herein. Mitochondrial dysfunction can occur as aresult of many factors. Non-limiting examples include mitochondrialdysfunction due to a disease (e.g., an age-related disease, amitochondrial disease or disorder, a neurodegenerative disease, aretinal disease, a genetic disease), diabetes, a hearing disorder, orany combination thereof. Mitochondrial dysfunction can involve thefunction of the endogenous mitochondria being reduced by greater than5%, greater than 10%, greater than 20%, greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, or greater than 90%. Therefore, in some embodiments, the endogenousmitochondria includes mitochondria with reduced function of about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

The methods provided herein are applicable to both homoplasmic andheteroplasmic mtDNA. In specific embodiments, the endogenous mtDNA is asingle type of mtDNA (i.e., the endogenous mtDNA is homoplastic). Inother specific embodiments, the endogenous mtDNA includes more than onetype of mtDNA (i.e., the endogenous mtDNA is heteroplasmic). In someembodiments, the heteroplasmic mtDNA includes both wild-type mtDNA andmutant mtDNA. Generally, the proportion of mutant mtDNA determines theseverity of the phenotype and can influence the degree to whichmitochondrial function is reduced. For example, in some embodiments theheteroplasmic mtDNA is 5% mutant mtDNA and 95% wild-type mtDNA, and themitochondrial function is reduced 5%. In other embodiments, theheteroplasmic mtDNA is 55% mutant mtDNA and 45% wild-type mtDNA, and themitochondrial function is reduced 55%. However, it is understood thatthe percentage of mutant mtDNA need not be proportional to themitochondrial function.

Dysfunctional mitochondria is generally characterized by a loss ofefficiency in the electron transport chain and reductions in thesynthesis of high-energy molecules, such as adenosine-5′-triphosphate(ATP), the leakage of deleterious reactive oxygen species (ROS), and/ordisrupted cellular respiration. A person skilled in the art wouldunderstand how to evaluate mitochondrial function. For example,cell-based assays, such as the Seahorse Bioscience XF Extracellular FluxAnalyzer, can used performed for the determination of basal oxygenconsumption, glycolysis rates, ATP production, and respiratory capacityin a single experiment to assess mitochondrial dysfunction. Similarly,the Oroboros 02K respirometer can also be used to establish quantitativefunctional mitochondrial diagnosis. It is understood that the assayexamples described above are exemplary and are not inclusive of allmethods to evaluate mitochondrial function.

In some embodiments, functional mitochondria have an intact outermembrane. In some embodiments, functional mitochondria are intactmitochondria. In another embodiment, functional mitochondria consumeoxygen at an increasing rate over time. In another embodiment, thefunctionality of mitochondria is measured by oxygen consumption. Inanother embodiment, oxygen consumption of mitochondria may be measuredby any method known in the art such as, but not limited to, theMitoXpress fluorescence probe (Luxcel). In some embodiments, functionalmitochondria are mitochondria which display an increase in the rate ofoxygen consumption in the presence of ADP and a substrate such as, butnot limited to, glutamate, malate or succinate. Each possibilityrepresents a separate embodiment of the present invention. In anotherembodiment, functional mitochondria are mitochondria that produce ATP.

While the methods provided herein can be useful in generating a MirCfrom a recipient cell that has dysfunctional mitochondrial, mutantmtDNA, or a combination thereof, it is also understood that the MirCgeneration need not be performed in a recipient cell with dysfunctionalmitochondria. In some embodiments, a MirC is generated using a recipientcell with functional endogenous mitochondria, wild-type mtDNA, or acombination thereof, and the exogenous mitochondria is also functional,contains wild-type mtDNA, or a combination thereof. For example,endogenous wild-type mtDNA can be reduced using the methods providedherein and exogenous wild-type mtDNA can be transferred into therecipient cell, such as mitochondrial replacement in an “old” recipientcell (e.g., a cell from an aged subject or a cell with relatively highpopulation doubling level (PDL)) with exogenous mtDNA from a healthydonor cell (e.g., a young cell with relatively low PDL). Thus, incertain embodiments, the exogenous mtDNA is from a donor cell that is ahealthy donor cell, for example a donor cell that is younger than therecipient cell. In certain embodiments, the donor and recipient cellhave a difference in PDL of about 1.5 fold, about 2 fold, about 2.5fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold.In other embodiments, the donor and recipient cells are from subjectsthat are separated in age by about 1.5 fold, about 2 fold, about 2.5fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold.However, it is understood that a difference in age between the donorcell and the recipient cell is not a requirement. In some embodiments,the donor and recipient cell are the same age, and the donor cell is aheathy cell.

In yet other embodiments, the generation of a MirC is performed in arecipient cell with functional endogenous mitochondria, such aswild-type endogenous mtDNA, and the exogenous mtDNA is mutant, encodesfor dysfunctional mitochondria, the exogenous mitochondria isdysfunctional, or a combination thereof. In other embodiments, theexogenous mitochondria, exogenous mtDNA, or a combination thereof isfrom a donor cell that is older than the recipient cell. For example, insome embodiments, a model of a mitochondrial disease or disorder can becreated by replacement of functional mitochondria in a recipient cellwith exogenous mtDNA from a donor cell that is mutant and/or encodes fordysfunctional mitochondria. It is understood that the examples describedherein are exemplary and are not inclusive of all combinations involvingmtDNA replacement.

As provided herein, the methods of generating a MirC can be practicedusing either an agent that reduces endogenous mtDNA, or an agent thatreduces endogenous mitochondrial function. In certain circumstances, acombination of the two agents can be used. Agents that are capable ofreducing mitochondrial function are well known in the field, and arewithin the skillset of a person skilled in the art. Exemplary agentsinclude inhibitors of the mitochondrial respiratory chain that blockrespiration in the presence of either ADP or uncouplers, such as aninhibitor of complex III (e.g., myxothiazol), an inhibitor of complex IV(e.g., sodium azide, potassium cyanide (KCN)), or an inhibitor ofcomplex V (e.g., oligomycin); inhibitors of phosphorylation that abolishthe burst of oxygen consumption after adding ADP, but have no effect onuncoupler-stimulated respiration; uncoupling agents that abolish theobligatory linkage between the respiratory chain and the phosphorylationsystem which is observed with intact mitochondria (e.g., dinitrophenol,CCCP, FCCP); ATP/ADP transport inhibitor, such as an adenine nucleotidetranslocase inhibitor (e.g., atractyloside) that either prevent theexport of ATP, or the import of raw materials across the mitochondrialinner membrane; ionophores (e.g. valinomycin, nigericin) which make theinner membrane permeable to compounds which are ordinarily unable tocross; or a Krebs cycle inhibitor (e.g. arsenite, aminooxyacetate) whichblock one or more of the TCA cycle enzymes, or an ancillary reaction. Itis understood that the agents that are capable of reducing mitochondrialfunction described above are non-limiting, and that a person skilled inthe art can readily identify suitable agents that are capable ofreducing mitochondrial function using techniques known in the art.

In specific embodiments, the agent that reduces endogenous mitochondrialfunction transiently reduces the endogenous mitochondrial function. Inother embodiments, the agent that reduces endogenous mitochondrialfunction permanently reduces the endogenous mitochondrial function. Inpreferred embodiments, the agent that reduces endogenous mitochondrialfunction partially reduces the endogenous mitochondrial function.

Various agents can be used to reduce mtDNA. In certain embodiments, theagent that reduces mtDNA is selected from a nucleic acid encoding afusion protein comprising a mitochondrial-targeted sequence (MTS) and anendonuclease, an endonuclease, or a small molecule. In certainembodiments, the small molecule is a nucleoside reverse transcriptaseinhibitor (NRTI). The nucleic acid can be a messenger ribonucleic acid(mRNA) or a deoxyribonucleic acid (DNA). In certain embodiments, theagent that reduces mtDNA is a plasmid DNA expression vector cassetteencoding an endonuclease. In preferred embodiments, the agent is aplasmid DNA expression vector cassette encoding an endonuclease with aMTS. Various expression vector cassettes can be used, and a personskilled in the art would understand the necessary considerationsrequired to enable successful expression of the endonuclease dependingon the host cell. For example, a mammalian expression vector, such as avector having a cytomegalovirus (CMV) promoter, SV40 promoter, or CAGpromoter, would be suitable for expression of the endonuclease in amammalian cell, but not a non-mammalian cell. Similarly, it isunderstood that viral expression vectors can also be used and a personskilled in the art would understand that such viral expression vectorsmay require helper plasmids (i.e., envelope and packaging plasmids) tobe used in tandem with the transfer plasmid. In other embodiments, theagent is an mRNA encoding an endonuclease. In other preferredembodiments, the agent is an mRNA encoding an endonuclease with a MTS.In yet further embodiments, the agent is an endonuclease that is arecombinant protein. In other embodiments, the agent is a smallmolecule, such as, for example, a small molecule that disrupts synthesisof mtDNA. Techniques for generating any of the expression methods areknown to those skilled in the art, and can be readily performed withoutundue experimentation. In preferred embodiments, the agent is suitablefor clinical use.

In specific embodiments, the endonuclease can be a restriction enzymethat cleaves DNA double helices into fragments at specific sites, suchas XbaI, which cleaves the following sequence of DNA:

The endonuclease can also include, for example, restriction enzymesother than XbaI, such as EcoRI, BamHI, HindIII, or PstI, which alldigest mtDNA at multiple sites. Endonucleases have defined recognitionsites, which allows prediction of their sensitivity on mtDNA. Thedefined recognition sites of restriction enzymes, such as, for example,XbaI, EcoRI and SmaI, are specific to a given nucleic acid sequence.Accordingly, in some embodiments, the reduction of endogenous mtDNA canbe performed using zinc fingers and transcription activator-likeeffectors (TALEs) that have been combined to DNA nucleases. These twotypes of DNA-binding proteins can be engineered to have specificitiesfor new DNA sequences of interest. Similarly, clustered regularlyinterspaced short palindromic repeats (CRISPR)/Cas9 proteins can be alsointroduced into cells through the addition of the corresponding encodinggenes. Therefore, in some embodiments, the endonuclease can be aprogrammable nuclease, such as a RNA-guided DNA endonuclease (e.g.,Cas9), zinc finger nuclease (ZFN), or transcription activator-likeeffector nuclease (TALEN). It is understood that the nucleases describedabove are non-limiting, and that a person skilled in the art can readilyidentify suitable endonucleases using techniques known in the art. Forexample, the Cambridge Reference Sequence or similar consensus sequencecan be used to identify suitable endonucleases that recognize the mtDNAsequence by, for example, an in silico analysis. In specificembodiments, the endonuclease cleaves a wild-type sequence of mtDNA. Inother embodiments, the endonuclease cleaves a mutant sequence of mtDNA.It is also understood that the agent that reduces endogenous mtDNA neednot be an endonuclease and that any agent capable of reducing mtDNA canbe employed, including an agent that inhibits the biosynthesis of mtDNA,such as ethidium bromide. Also contemplated herein, are agents, such as,for example, Urolithin A or the small molecule p62-mediated mitophagyinducer (PMI), that induce autophagy in order to promote the selectivedegradation of endogenous mitochondria (i.e., mitophagy agonist). Thepresent invention can also be practiced using a nucleoside reversetranscriptase inhibitor (NRTI) as an agent that reduces mtDNA.

In addition, in some embodiments, the expression vector cassette caninclude one or more antibiotic resistance genes to enable selection of apopulation of cells that express the expression vector cassette. Forexample, in some embodiments, the expression vector can include thepuromycin N-acetyl-transferase gene (pac) from Streptomyces, and cellscan be selected using puromycin. In circumstances where selection isperformed using antibiotics, e.g., puromycin, the selection can be brief(e.g., 24-48 hours) to limit long term exposure to the drug. However, itis understood that the example provided above is merely exemplary, andthat the expression vector cassette can include other antibioticsresistance genes, such as, for example, the bsr, bls, or BSD gene forselection with Blasticidin, or the hph gene for selection withhydromycin B. It is generally understood that the concentration ofantibiotic used for selection will depend on the type of antibiotic andthe cell type, and would be readily obtainable to one skilled in the artwithout undue experimentation. It is further understood that selectioncan be produced by any means known in the art, and need not involveantibiotic resistance. For example, in some embodiments, selection ofthe cells can be performed by, for example, fluorescence-activated cellsorting (FACS) of a cell surface marker or expression of a fluorescentprotein encoded by the expression. In yet further embodiments, selectioncan be performed according to the cell's phenotype. For example, in someembodiments, the successful deletion of mutant endogenous mtDNA in acell with heteroplasmy can result in a phenotypic response that isselectable, such as, for example, cell survival.

Accordingly, in some embodiments, the cells are selected afterintroducing an expression vector cassette that contains an endonucleasethat degrades mtDNA. In some embodiments, the cells are selected toobtain a homogenous population of cells that express an endonucleasethat degrades mtDNA. In specific embodiments, the cells are selectedafter introducing an expression vector cassette that contains anendonuclease that degrades mtDNA, and a homogenous, stable cell line isgenerated. In other embodiments, the cells are selected to enrich for apopulation of cells that express an endonuclease that degrades mtDNA. Asdescribed above, this enrichment by selection can involve a briefexposure to an antibiotic. The enriched cells can stably express theendonuclease or transiently express the endonuclease depending on theextent and/or manner of the selection pressure. It is understood that anenriched population need not be homogenous, and that an enrichedpopulation of cells that express an endonuclease that degrades mtDNAcontains a higher percentage of cells with the endonuclease, relative toan unselected population of cells, but may also contain some cells thatdo not express the endonuclease.

In other embodiments, the cells are not selected after introducing anexpression vector that contains an endonuclease that degrades mtDNA. Inspecific embodiments, the cells are not selected after introducing anexpression vector that contains an endonuclease that degrades mtDNA, andthe endonuclease is transiently expressed.

Various methods for introducing the plasmid DNA expression vectorcassette, mRNA, and/or recombinant protein are known in the art. In someembodiments, the plasmid DNA expression vector cassette is introduced byelectroporation. In specific embodiments, the electroporation method isflow electroporation, such as MaxCyte Flow Electroporation. In otherspecific embodiments, the electroporation method includes thenucleofection technology, such as Lonza's Nucleofector™ technology. Inother embodiments, the plasmid DNA expression vector cassette isintroduced by cationic lipid transfection. In yet further embodiments,the plasmid DNA expression vector cassette is introduced by viraltransduction. It is understood that the methods described above forintroducing the expression vector cassette are non-limiting and merelyintended to be exemplary methods, and that any method known in the artcan be used for introducing the DNA expression vector cassette.

Where the agent for reducing endogenous mitochondria comprises anendonuclease, expression of the endonuclease can also involveintroduction of mRNA encoding the endonuclease or introducing theendonuclease as a recombinant protein. In certain embodiments, theMaxCyte electroporator can be used for mRNA transfection, particularlyin the clinical setting, which has cleared the standards of GoodManufacturing Practice and Good Clinical Practice. The transfection canbe performed using the MaxCyte electroporator according to themanufacturer's protocol. It is further understood that the methodsdescribed above are merely exemplary and that any means of introducingmRNA and/or recombinant protein can be used.

The specific targeting of the endonuclease to the mitochondria can beperformed by incorporating a mitochondrial targeting sequence (MTS)adjacent to the endonuclease coding sequence, which will result in afusion protein that targets the mitochondria. Strong MTSs have beenidentified and shown capable of targeting proteins to specificcompartments when fused on their N-termini, and are termed mitochondrialtargeting sequences. MTS suitable for the methods of the presentinvention are well known to the person skilled in the art (see, e.g.,U.S. Pat. No. 8,039,587B2, which is hereby incorporated by reference inits entirety). For example, MTS to the mitochondrial matrix can be used,such as the MTS that is a targeting peptide from the cytochrome coxidase subunit IV (COX 4), subunit VIII (COX 8), or subunit X (COX 10).In principle, any target sequence derived from any nuclear encodedmitochondrial matrix or inner membrane enzyme or an artificial sequencethat is capable of rendering the fusion protein into a mitochondrialimported protein (hydrophobic moment greater than 5.5, at least twobasic residues, amphiphilic alpha-helical conformation; see, e.g.,Bedwell et al., Mol Cell Biol. 9(3) (1989), 1014-1025) is useful for thepurposes of the present invention.

In certain embodiments, the MTS is a human MTS. In another embodiment,the MTS is from another species. Non-limiting examples of such sequencesare the cytochrome c oxidase subunit X (COX 10) MTS(MAASPHTLSSRLLTGCVGGSVWYLERRT, SEQ ID NO: 36), and the cytochrome coxidase subunit VIII (COX 8) MTS (MSVLTPLLLRSLTGSARRLMVPRA, SEQ ID NO:37). Additional non-limiting examples of MTS sequences are the naturalMTS of each individual mitochondrial protein that is encoded by thenuclear DNA, translated (produced) in the cytoplasm and transported intothe mitochondria, as well as citrate synthase (cs), lipoamidedeydrogenase (LAD), and C6ORF66 (ORF). The various MTS may beexchangeable for each mitochondrial enzyme among themselves.Accordingly, in some embodiments, the MTS targets a mitochondrial matrixprotein. In specific embodiments, the mitochondrial matrix protein issubunit VIII of human cytochrome C oxidase. Each possibility representsa separate embodiment of the fusion protein for use of the presentinvention.

Upon contacting a recipient cell with an agent that reduces endogenousmtDNA copy number or an agent that reduces endogenous mitochondrialfunction, the recipient cell is incubated for a sufficient period oftime for the agent to partially reduce the endogenous mtDNA copy numberin the recipient cell or partially reduce the endogenous mitochondrialfunction in the recipient cell, respectively. Identifying the“sufficient period of time” to allow the agent to reduce partiallyreduce the endogenous mtDNA copy number or partially reduce theendogenous mitochondrial function is within the skill of those in theart. The sufficient or proper time period will vary according to variousfactors, including but not limited to, the particular type of cells, theamount of starting material (e.g., the number of recipient cells and/oramount of mtDNA to be reduced), the amount and type of agent(s), theplasmid promoter regulator(s), and/or the culture conditions. In variousembodiments the sufficient period of time to allow a partial reductionof the endogenous mtDNA copy number in a recipient cell is about 1 day,about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,about 7 days, about 8 days, about 9 days, about 10 days, about 1-2weeks, about 2-3 weeks or about 3-4 weeks. In preferred embodiments, thesufficient period of time will be long enough that the resultingrecipient cell has a reduction in a majority of the endogenous mtDNAcopy number or a reduction in the function of a majority of theendogenous mitochondria and is also substantially free of the agent thatreduces endogenous mtDNA or the agent that reduces endogenousmitochondrial function before incubating the recipient cell with anexogenous mtDNA and/or exogenous mitochondria.

An important and novel aspect of the present invention is the findingthat mitochondrial transfer efficiency is severely reduced in cells witha complete depletion of endogenous mitochondria (i.e., (ρ) 0 cells), butcan be greatly improved when the endogenous mtDNA copy number is reducedbut not completely depleted (i.e., (ρ)⁻ cells). Furthermore, the presentinvention also demonstrates that simple add-on or centrifugationprotocols are inefficient without partial reduction in the endogenousmtDNA copy number. Accordingly, in preferred embodiments, the reductionof the endogenous mtDNA copy number in the recipient cell is less than a100% depletion of the endogenous mtDNA. In some embodiments, theendogenous mtDNA copy number in the recipient cell is reduced by about5% to about 99%. In specific embodiments, the agent that reducesendogenous mtDNA copy number reduces about 30% to about 70% of theendogenous mtDNA copy number. In other embodiments, the agent thatreduces endogenous mtDNA copy number reduces about 50% or more, about60% or more, about 70% or more, about 80% or more, or about 90% or more,or about 95% or more of the endogenous mtDNA copy number. In yet furtherembodiments, the agent that reduces endogenous mtDNA copy number reducesabout 60% to about 90% of the endogenous mtDNA copy number. It is alsounderstood that in some embodiments, the agent that reduces endogenousmtDNA copy number reduces mitochondrial mass.

In certain embodiments, the exogenous mtDNA is contained in isolatedexogenous mitochondria from a donor cell. Mitochondrial isolation may beaccomplished by any of a number of well-known techniques including butnot limited to those described herein, and in the cited references. Incertain embodiments, the exogenous mitochondria for use in mitochondrialtransfer is isolated using a commercial kit, such as, for example, theQproteum mitochondria isolation kit (Qiagen, USA), the MITOISO2mitochondria isolation kit (Sigma, USA), or Mitochondria Isolation Kitfor Cultured Cells (Thermo Scientific). In other embodiments, theexogenous mitochondria for use in mitochondrial transfer is isolatedmanually. For example, an exemplary manual isolation of mitochondriaincludes isolating the mitochondria from donor cells by pelleting thedonor cells, washing the cell pellet of 1-2 mL derived fromapproximately 10⁹ cells grown in culture, swelling the cells in ahypotonic buffer, rupturing the cells with a Dounce or Potter-Elvehjemhomogenizer using a tight-fitting pestle, and isolating the mitochondriaby differential centrifugation. Manual isolation can also include, forexample, sucrose density gradient ultracentrifugation, or free-flowelectrophoresis. Without wishing to be bound by any particular method,it is understood that the kits and manual methods described herein areexemplary, and that any mitochondrial isolation method can be used, andwould be within the skill set of a person skilled in the art.

In some embodiments, the isolated donor mitochondria is substantiallypure of other organelles. In other embodiments, the isolatedmitochondria can contain impurities and is enriched for mitochondria.For example, in some embodiments, the isolated mitochondria are about90% pure, about 80% pure, about 70% pure, about 60%, pure, about 50%,pure, or any integer in-between. In general, it is understood that anyimpurities contained with the isolated donor mitochondria will notaffect the viability or function of the recipient cell uponmitochondrial transfer. In specific embodiments, the transfer of theexogenous mitochondria, exogenous mtDNA, or a combination thereof doesnot involve transfer of non-mitochondrial organelles.

The quantity and quality of isolated mitochondria can easily bedetermined by a number of well-known techniques including but notlimited to those described herein, and in the cited references. Forexample, in some embodiments, the quantity of isolated mitochondria isdetermined by assessment of total protein content. Various methods areavailable for measurement of total protein content, such as the Biuretand Lowry procedures (see, e.g., Hartwig et al., Proteomics, 2009 June;9(11):3209-14). In other embodiments, the quantity of isolatedmitochondria is determined by mtDNA copy number.

In some embodiments, the isolated mitochondria are functionalmitochondria. In further embodiments, the isolated mitochondria aredysfunctional mitochondria. In some embodiments, the mitochondrialfunction can be assessed in the donor cell prior to isolation. In otherembodiments, the mitochondrial function can be assayed from the isolatedmitochondria.

The preservation of mitochondrial membrane integrity is anotherimportant factor during mitochondria isolation. In some embodiments, themtDNA used in the methods provided herein is from intact mitochondria.In specific embodiments, about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or greater than90% of the isolated mitochondria are intact. Mitochondrial membraneintegrity can be accomplished by any of a number of well-knowntechniques including but not limited to those described herein, and inthe cited references. For example, TMRM, Rhod123, JC-1 and DiOC6 aretypical probes for measurement of mitochondrial membrane potential (see,e.g., Perry et al., Biotechniques, 2011 February; 50(2):98-115). JC-1 isa widely used dye for measurement of inner-membrane potential ofisolated mitochondria, and is based on electrochemical proton gradientof mitochondrial inner membrane.

In certain embodiments of the methods provided herein, the recipienthaving a partial reduction of endogenous mtDNA in co-incubated withexogenous mitochondria from a healthy donor for a sufficient period oftime to non-invasively transfer exogenous mitochondria into therecipient cell, thereby generating a mitochondria replaced cell. Inother embodiments, the recipient having a partial reduction ofendogenous mtDNA in co-incubated with exogenous mtDNA from a healthydonor for a sufficient period of time to non-invasively transferexogenous mitochondria into the recipient cell, thereby generating amitochondria replaced cell. Identifying the “sufficient period of time”to non-invasively transfer exogenous mitochondria and/or exogenous mtDNAinto the recipient cell is within the skill of those in the art. Thesufficient or proper time period will vary according to various factors,including but not limited to, the particular type of cells, the amountof starting material (e.g., the number of recipient cells and/or amountof endogenous mtDNA to be replaced), the amount of donor material (e.g.,the quantity, quality, and/or purity of exogenous mtDNA) and/or theculture conditions. In various embodiments the sufficient period of timeto non-invasively transfer exogenous mitochondria and/or exogenous mtDNAinto a recipient cell is about 1 day, about 2 days, about 3 days, about4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 1-2 weeks, about 2-3 weeks or about 3-4weeks. In certain embodiments, at the end of the co-incubation period,the recipient cells will have a majority of the exogenous mtDNA and besubstantially free of any exogenous mitochondria organelles.

Another feature of the current invention is the finding that the totalmtDNA copy number in the MirC does not substantially increase, relativeto the original recipient cell. In contrast, other less efficientmethods have attempted to add on mitochondria without modulating therecipient cell before the co-incubation step, or transfer exogenousmitochondria using centrifugation without modulating the recipient cellprior to the centrifugation. Consequently, the resultant cellpopulations using the inefficient methods tend to have large increase inthe total mtDNA copy number. Thus, in certain embodiments, themitochondria replaced cell has a total mtDNA copy number no greater thanabout 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about1.5 fold, or more, relative to the total mtDNA copy number of therecipient cell prior to contacting with the agent that reducesendogenous mtDNA copy number.

The use of non-invasive transfer is another unique aspect of the presentinvention. Previous methods have employed invasive instruments to injectexogenous mitochondria, physically force the mitochondria into the cellsby centrifugation, or similar harsh conditions that are harmful to therecipient cells. In clinical settings, particularly when the recipientcell number may be limited, such as with hematopoietic stem cells or Tcells, harsh manipulation of the cells in undesirable. Therefore, theuse of the non-invasive transfer is a beneficial feature of thisinvention, which lends itself to use in the clinical setting.

As provided herein, the exogenous mitochondria, exogenous mtDNA, or acombination thereof can be autologous or allogeneic to the recipientcell. In some embodiments, the exogenous mtDNA is allogeneic, relativeto the recipient cell. For example, the exogenous mtDNA can be obtainedfrom the same species as the recipient cell, and have a differentgenotype than that of the recipient cell. In other embodiments, theexogenous mitochondria, exogenous mtDNA, or a combination thereof isautologous. By way of example, an exemplary autologous exogenous mtDNAcan include mtDNA from a healthy donor cell, for example a “young” donorcell such as from umbilical cord blood, and the recipient cell can befrom the same subject, and be an “old” recipient cell, where the terms“young” and “old” refer to the total number of times the cells in thepopulation have doubled or the age of the subject from which the cellsare taken. Another exemplary autologous exogenous mtDNA can include, forexample, donor mtDNA that has been isolated from the same subject as therecipient cell and modified prior to replacing it with the recipientcell. In certain embodiments, only the mtDNA and/or mitochondria areallogenic and the recipient cell is autogenic to the subject in need ofan exogenous mtDNA and/or exogenous mitochondria.

In certain embodiments, the replacement of mtDNA in the recipient cellcan be evaluated by sequencing the DNA sequences of hyper variableregion (HVR) of mtDNA, for example, the HV1 and/or HV2 of the D-loop,and comparing it to the sequence of both the donor mitochondria and therecipient cells. In specific embodiments, the differences in sequencesbetween the recipient cell and the donor mitochondria can be identifiedby a Single Nucleotide Polymorphism assay. For example, the amplifiedsequences of the mtDNA from the recipient cell and the donormitochondria can be cloned into a plasmid for use as a standard forquantification.

In some embodiments, the cells (i.e. donor cells and recipient cells)are animal cells or plant cells. In specific embodiments, the cells aremammalian cells. In some embodiments, the cells are isolated from amammalian subject who is selected from a group consisting of: a human, ahorse, a dog, a cat, a mouse, a rat, a cow and a sheep. In someembodiments, the cells are human cells. In some embodiments, the cellsare cells in culture. The cells may be obtained directly from a mammal(preferably human), or from a commercial source, or from tissue, or inthe form for instance of cultured cells, prepared on site or purchasedfrom a commercial cell source and the like. In certain embodiments, thecells are primary cells (i.e., cells obtained directly from livingtissue, for example, biopsy material). The cells may come from any organincluding, but not limited to, the blood or lymph system, from muscles,any organ, gland, the skin, or the brain. In certain embodiments, thecells are somatic cells. In some embodiments, the cells are selectedfrom the group consisting of epithelial cells, neural cells, epidermalcells, keratinocytes, hematopoietic cells (e.g., bone marrow cells),melanocytes, chondrocytes, hepatocytes, B-cells, T cells, erythrocytes,macrophages, monocytes, fibroblasts, muscle cells, vascular smoothmuscle cells, hepatocytes, splenocytes, and pancreatic_(R) cells.

As provided herein, in specific embodiments, the donor cells arecommercially available cells cultured under Current Good ManufacturingPractices (cGMP). For example, the donor cells can be obtained from acell repository, such as Waisman Biomanufacturing, or similar commercialresource, such as a commercial source that generates cGMP compliantcells. In some embodiments, the donor cells are cGMP manufacturedbone-marrow derived Mesenchymal Stromal Cells (BM-MSCs). In otherembodiments, the cells are cGMP grade human hepatocytes. As such, it isalso understood that the donor cells can be frozen cells that are thawedprior to isolating the mitochondria. However, the mitochondria need notbe isolated after freezing the cells, and can be isolated from freshcells and used immediately, or, in certain embodiments, the mitochondriacan be isolated and then frozen before transferring into the recipientcell.

In some embodiments, the cells are cancer cells. Typically, the cancercells are isolated from a cancer selected from the group consisting ofbreast cancer, prostate cancer, lymphoma, skin cancer, pancreaticcancer, colon cancer, melanoma, malignant melanoma, ovarian cancer,brain cancer, primary brain carcinoma, head-neck cancer, glioma,glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer,head or neck carcinoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma,testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomachcarcinoma, colon carcinoma, prostatic carcinoma, genitourinarycarcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiplemyeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,adrenal cortex carcinoma, malignant pancreatic insulinoma, malignantcarcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia,acute granulocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma.

In some embodiment, the cells are stem cells. As used herein, the term“stem cell” refers to an undifferentiated cell that can be induced toproliferate. The stem cell is capable of self-maintenance orself-renewal, meaning that with each cell division, one daughter cellwill also be a stem cell. Stem cells can be obtained from embryonic,post-natal, juvenile, or adult tissue. Stem cells can be pluripotent ormultipotent. The term “progenitor cell,” as used herein, refers to anundifferentiated cell derived from a stem cell, and is not itself a stemcell. Some progenitor cells can produce progeny that are capable ofdifferentiating into more than one cell type. Stem cells includepluripotent stem cells, which can form cells of any of the body's tissuelineages: mesoderm, endoderm and ectoderm. Therefore, for example, stemcells can be selected from a human embryonic stem (ES) cell; a humaninner cell mass (ICM)/epiblast cell; a human primitive ectoderm cell, ahuman primitive endoderm cell; a human primitive mesoderm cell; and ahuman primordial germ (EG) cell. Stem cells also include multipotentstem cells, which can form multiple cell lineages that constitute anentire tissue or tissues, such as but not limited to hematopoietic stemcells or neural precursor cells. Stem cells also include totipotent stemcells, which can form an entire organism. In some embodiment, the stemcell is a mesenchymal stem cell. The term “mesenchymal stem cell” or“MSC” is used interchangeably for adult cells which are not terminallydifferentiated, which can divide to yield cells that are either stemcells, or which, irreversibly differentiate to give rise to cells of amesenchymal cell lineage, e.g., adipose, osseous, cartilaginous, elasticand fibrous connective tissues, myoblasts) as well as to tissues otherthan those originating in the embryonic mesoderm (e.g., neural cells)depending upon various influences from bioactive factors such ascytokines. In some embodiments, the stem cell is a partiallydifferentiated or differentiating cell. In some embodiments, the stemcell is an induced pluripotent stem cell (iPSC), which has beenreprogrammed or de-differentiated. In specific embodiments, therecipient cell is an iPSC. In other embodiments, the recipient cell is ahematopoietic stem cell (HSC) or a MSC. Stem cells can be obtained fromembryonic, fetal or adult tissues.

In other embodiments, the cells are immune cells. In specificembodiments, the recipient cell is an immune cell. In some embodiments,the immune cell is selected from the group consisting of a T cell, aphagocyte, a microglial cell, and a macrophage. In specific embodiments,the T cell is a CD4+ T cell. In other embodiments, the T cell is a CD8+T cell. In yet further embodiments, the T cell is a chimeric antigenreceptor (CAR) T cell. In specific embodiments, the recipient cell is anexhausted or near exhausted T cell in a state or near a state of T celldysfunction.

5.3 Method of Enhanced Mitochondrial Transfer

Also provided herein are methods for the transfer of mtDNA and/ormitochondria that involve the use of a second active agent incombination with any of the methods described in Section 5.2. Thetransfer of mitochondria has been reported to involve the endocytosispathway, which is an ATP-dependent process. For example, under certaincell culture conditions, mitochondria have been observed to be engulfedvia macropinocytosis (see, e.g., Kitani et al., J Cell Mol Med., 2014,18(8):1694-1703). Accordingly, the present invention also relates to thenovel findings that the use of a second active agent prior toco-incubating the recipient cell with exogenous mitochondria and/orexogenous mtDNA can promote the uptake of the exogenous mitochondriaand/or exogenous mtDNA.

Various types of agents can be used to promote the uptake of theexogenous mitochondria and/or exogenous mtDNA. In some embodiments, thesecond active agent is selected from the group consisting of largemolecules, small molecules, or cell therapies, and the second activeagent is optionally selected from the group consisting of rapamycin, NR(Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate(RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176,Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone),mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin,ketogenic treatment, hypoxia, and an activator of endocytosis. Inspecific embodiments, the activator of endocytosis is a modulator ofcellular metabolism. Cellular metabolism can be modulated using variousmethods known to one skilled in the art. In certain embodiments,modulation of cellular metabolism comprises nutrient starvation, achemical inhibitor, or a small molecule.

As described above, transfer of intact mitochondria has been reported tooccur by an endocytosis pathway. For example, the exogenous mitochondriaand/or exogenous mtDNA can be transferred by uptake of intactmitochondria via the endocytosis pathway. The endocytosis pathways canbe subdivided into four categories: 1) clathrin-mediated endocytosis, 2)caveolae, 3) macropinocytosis, and 4) phagocytosis. Clathrin-mediatedendocytosis is mediated by small (approx. 100 nm in diameter) vesiclesthat have a morphologically characteristic coat made up of a complex ofproteins that are mainly associated with the cytosolic protein clathrin.Accordingly, in certain embodiments, the endocytosis pathway formitochondrial transfer is a clathrin-dependent endocytosis pathway. Inother embodiments, the endocytosis pathway for mitochondrial transfer isa clathrin-independent pathway. In specific embodiments, the endocytosispathway is macropinocytosis.

Macropinocytosis has been suggested to be an important process innutrient-deprived environments. As a result, it was hypothesized that ashortage of cellular nutrients or an inhibition of the pathways ortarget molecules that are activated with sufficient nutrition such asmTOR could be a strategy to augment the cellular engulfment of intactmitochondria into the cytosol. Specifically, as provided herein, it wasdiscovered that a suppression of mTOR can enhance the uptake ofexogenous mitochondria. mTOR is an essential sensor of amino acids,energy, oxygen, and growth factors, and a key regulator of protein,lipid, and nucleotide synthesis that is involved in uptake ofextracellular nutrients. Accordingly, in some embodiments, the methodsprovided herein further comprise contacting a recipient cell with asmall compound, a peptide, or a protein that can increasemacropinocytosis. In specific embodiments, the methods provided hereinfurther comprise modulating cellular metabolism of a recipient cellprior to transfer of the exogenous mitochondria and/or exogenous mtDNA.In certain embodiments, the modulating cellular metabolism is performedusing the same small compound, a peptide, or a protein that can increasemacropinocytosis.

Modulating cellular metabolism may be accomplished by any of a number ofwell-known techniques including but not limited to those describedherein, and in the cited references. For example, in some embodiments,modulating cellular metabolism is performed by nutrient starvation ornutrient deprivation. In other embodiments, modulating cellularmetabolism is performed by a chemical inhibitor or small molecule. Inspecific embodiments, the chemical inhibitor or small molecule is anmTOR inhibitor.

Various compounds are known to inhibit mTOR, including rapamycin, alsoknown as sirolimus (CAS Number 53123-88-9; C₅₁H₇₉NO₁₃), and rapamycinderivatives (e.g., rapamycin analogs, also known as “rapalogs”).Rapamycin derivatives include, for example, temsirolimus (CAS Number162635-04-3; C₅₆H₈₇NO₁₆), everolimus (CAS Number 159351-69-6;C₅₃H₈₃NO₁₄), and ridaforolimus (CAS Number 572924-54-0; C₅₃H₈₄NO₁₄P).Accordingly, in some embodiments, the methods provided herein formitochondrial transfer further comprise modulating cellular metabolismof a recipient cell prior to transfer of the exogenous mitochondriaand/or exogenous mtDNA using rapamycin or a derivative thereof. It isunderstood that the embodiments described above for modulating cellularmetabolism are non-limiting, and modulating cellular metabolism need notinvolve a chemical compound or small molecule.

Accordingly, in some embodiments, rapamycin or a derivative thereof,which include clinically approved drugs, can be utilized to increase thetransfer efficiency of exogenous mitochondria, either as a stand-alonemethod or in combination with any of the methods provided herein, suchas methods involving the partial reduction in the endogenousmitochondria of the recipient cells.

One skilled in the art would understand that additional methods ofdelivery can also be used to introduce the exogenous mitochondria and/orexogenous mtDNA, and that macropinocytosis is an exemplary pathway. Insome embodiments, the mtDNA can be delivered by clathrin-dependentendocytosis, or clathrin-independent endocytosis. In specificembodiments, the clathrin-independent pathway can be, for example,CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis,flotillin-dependent endocytosis, macropinocytosis, circular doralruffles, phagocytosis, or trans-endocytosis. It is further understoodthat delivery of exogenous mitochondria and/or exogenous mtDNA can beenhanced by the use of any compound that stimulates mitochondrialdelivery, such as an activator of endocytosis. Non-limiting exemplarycompounds suitable for activating endocytosis include, for example,phorbol-12-myristate-13-acetate (PMA) (C₃₆H₅₆O₈),12-O-tetradecanoylphorbol 13-acetate (TPA) (C₃₆H₅₆O₈), tanshinone IIAsodium sulfonate (TSN-SS) (C₁₉H₁₇O₆S.Na), and phorbol-12,13-dibutyrate,or derivatives thereof. Furthermore, it is also understood thatnon-endocytosis mediated transfer of mtDNA and/or mitochondria can beused, including methods that bypass endocytosis and/or cell fusion.

5.4 Methods of Treatment

Provided herein are various methods for the treatment of conditionsassociated with mutant mtDNA and/or dysfunctional mitochondria, uses ofcompositions for the treatment of conditions associated with mutantmtDNA and/or dysfunctional mitochondria, and uses of compositions in themanufacture of medicaments for the treatment of conditions associatedwith mutant mtDNA and/or dysfunctional mitochondria. Also provided aremethods of treatment involving the use of exogenous mitochondria and/orexogenous mtDNA to restore or enhance the function of endogenousmitochondria, uses of compositions to restore or enhance the function ofendogenous mitochondria, and uses of compositions in the manufacture ofmedicaments for the treatment of a subject in need of mitochondrialreplacement. In certain embodiments, the treatment involves preventionof mitochondrial dysfunction.

5.4.1 Methods of Treating an Age-Related Disease

In certain embodiments, provided herein are methods of treating asubject having or suspected of having an age-related disease involvingany of methods described in Section 5.2 and/or Section 5.3. In someembodiments, provided herein are methods of treating a subject having orsuspected of having an age-related disease involving generating amitochondria replaced cell ex vivo or in vitro by contacting a recipientcell with an agent that reduces endogenous mtDNA or reduces endogenousmitochondrial function, incubating the recipient cell for a sufficientperiod of time for the agent to partially reduce the mtDNA copy numberin the recipient cell or partially reduce the endogenous mitochondrialfunction, co-incubating (1) the recipient cell in which the endogenousmtDNA or endogenous mitochondrial function has been partially reduced,and (2) exogenous mitochondria and/or exogenous mtDNA from a healthydonor, for a sufficient period of time to non-invasively transferexogenous mitochondria into the recipient cell, thereby generating amitochondria replaced cell, and then administering a therapeuticallyeffective amount of the mitochondria replaced recipient cell to thesubject having or suspected of having an age-related.

In certain embodiments, the age-related disease includes an autoimmunedisease, a metabolic disease, a genetic disease, cancer, aneurodegenerative disease, and immunosenescence. The metabolic diseasecan include diabetes. Non-limiting examples of neurodegenerativediseases that can be treated by the methods provided herein includeAlzheimer's disease, or Parkinson's disease. In addition, the geneticdisease capable of being treated include Hutchinson-Gilford ProgeriaSyndrome, Werner Syndrome, and Huntington's disease. Additionalage-related diseases that involve dysfunctional mitochondria are alsocontemplated.

In certain embodiments, the methods of treating a subject having orsuspected of having an age-related disease involves generating a MirC,where the recipient cell used to generate the MirC is a T cell or ahematopoietic stem cell (HSC). For example, endogenous mtDNA, endogenousmitochondria, or a combination thereof in a senescent T cell orhematopoietic stem cell (HSC) can be replaced for rejuvenation. The invitro or ex vivo mitochondrial replacement can be a feasible option forthe treatment using human T cells and/or hematopoietic stem cells withdiseased patients. Thus in some embodiments, the methods provided hereincan be used to delay senescence and/or extending lifespan in a cell bynon-invasively transferring isolated exogenous mitochondria from ahealthy, non-senescent cell into a senescent or near senescent cell torejuvenate the recipient cell, and the resulting rejuvenated MirC canthen be administered to a patient having or suspected of having anage-related disease.

As demonstrated herein, the rejuvenation of senescent T cells is onepossible embodiment by which the present invention can be used to treata subject having an age-related disease, such as cancer. By way ofexample, an old T cell, exhibiting a Senescence Associated SecretoryPhenotype (SASP) consisting of inflammatory cytokines, growth factors,and proteases, reduced and/or slower rates of cell population doublings,shortened telomeres, increased DNA damage response (DDR), or acombination thereof can be rejuvenated by using the methods providedherein to non-invasively transfer, for example, isolated mitochondriafrom a young, healthy T cell that is autologous to a subject having anage-related disease, such as cancer. The T cell-derived MirC withcharacteristics of a young, non-senescent cell can then be administeredto the subject for treatment of the age-related disease.

Thus, in specific embodiments, the methods of treating a subject havingor suspected of having an age-related disease involves generation of aMirC where the recipient cell is a T cell. T cell fate is regulated bythe metabolic pathway, with either glycolysis or oxidativephosphorylation (OXPHOS) being responsible for providing a majority ofthe energy to T cells. Glycolysis dominant T cells select todifferentiate into effector T cells, whereas OXPHOS dominant T cells formemory T cells. Thus, exogenous mitochondria and/or mtDNA can be used tomodulate T cell fate. For example, in the case of allergy, exogenousmitochondria and/or mtDNA could be used to calm hyper-activated T cells.In other situations, such as in cancer immunotherapy, exogenousmitochondria and/or mtDNA could empower anti-tumor T cells to allow theT cells to persist for a longer time, or facilitate T cell lyticcapacity and/or reduce tumor burden. Moreover, emerging treatments usingchimeric antigen receptor T cells (CAR T) utilize autologous T cells.Those CAR T cells might be in fatigue due to aging or malnutrition suchas cachexia which is frequently seen in a severe pathologic stage ofcancer. The mitochondrial replacement technology may energize andrejuvenate CAR T to provide more ATP leading to better outcomes.

Accordingly, in certain embodiments, the methods of treating a subjectinvolve a recipient cell that is a T cell. The T cell can be a CD4+ Tcell, a CD8+ T cell, or a CAR T cell. In specific embodiments, themitochondrial replacement in the recipient T cell results in a T cellwith a prolonged lifespan. For example, the lifespan can be increasedabout 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold,or greater than 5 fold. In specific embodiments, the mitochondrialreplacement in the recipient T cell inhibits or delays senescence of therecipient T cells, as compared to a T cell without mitochondrialreplacement. As described in Section 5.2, lifespan can be prolonged byperforming mtDNA replacement using exogenous mitochondria and/orexogenous mtDNA from a donor cell that is younger than the recipientcell. In certain embodiments, the donor and recipient cell have adifference in PDL of about 1.5 fold, about 2 fold, about 2.5 fold, about3 fold, about 4 fold, about 5 fold, or greater than 5 fold. In otherembodiments, the donor and recipient cells are from subjects that areseparated in age by about 5 years, about 10 years, about 15 years, about20 years, or greater than 20 years. In other specific embodiments, themitochondrial replacement in the recipient T cell results in T cellshaving increased lytic capacity, relative to T cells not havingmitochondrial replacement. In yet further embodiments, the mitochondrialreplacement in the T cells results in reduced tumor burden.

Although in certain embodiments plasmid-based gene transfection can beused to generate a T cell with exogenous mitochondria and/or exogenousmtDNA, in other embodiments mRNA transfection can be used. The use ofmRNA transfection can decrease the chance of the RNA sequence beingintegrated into the host genome, and can also have minimal long-termgene expression that would cause the endogenous mtDNA reduction.

In certain embodiments, the MaxCyte electroporator can be used for mRNAtransfection, particularly in the clinical setting, which has clearedthe standards of Good Manufacturing Practice and Good Clinical Practice.The transfection can be performed using the MaxCyte electroporatoraccording to the manufacturer's protocol.

The methods of treating a subject having or suspected of having anage-related disease can also involve the generation of a MirC using themethods provided herein, where the recipient cell is a hematopoieticstem cell (HSC). Hematopoietic stem cells (HSC) supply not only bloodcells, but also, for example, the endothelium which fixes damagedresident cells in the remote organs via trans-differentiation. Moreover,malfunctions of HSCs have been reported to be involved in senescencethroughout the whole body. Therefore, it is contemplated thatHSC-derived MirC can be used as a method of treatment in any age-relateddisease.

In addition, allogenic HSC transplantation can result in rejection ofthe transplant or even graft-versus-host disease. Autologous HSCtransplantation is often a safer and more practical measure for diseaseintervention. For example, autologous HSC transplantation typically doesnot require the preconditioning with immunosuppressive agents, such asradiation and chemicals. Accordingly, in vitro or ex vivo generation ofa MirC using exogenous mtDNA from a healthy young mitochondria in anautologous HSC that is then returned back to the patients' bodies isenvisioned using the methods provided herein.

In certain embodiments, the HSC is autologous to the subject in need ofmitochondrial and/or mtDNA replacement, and the exogenous mtDNA isallogenic. As provided herein, the mtDNA replacement in an HSC canresult in a differentiated cell with functional mitochondria and/or adifferentiated cell with improved function. Accordingly, the methodsprovided herein can be used in the setting of HSC transplantation.

Aging alters the biological processes and leads to development ofdegenerative disorders, such as Alzheimer's disease, atherosclerosis,osteoporosis, type 2 diabetes mellitus, and tissue fibrosis which iscausative for chronic kidney disease and chronic obstructive pulmonarydisease. Mitochondria can play a role in senescence, via reactive oxygenspecies generated by mitochondria, which can impact the ageing process.Mitochondrial dysfunction in aging is in a vicious cycle related to aderegulated nutrient sensing where a shortage of nicotinamide adeninedinucleotide (NAD⁺), caused by downregulation of nicotinamidephosphoribosyltransferase (NAMPT) and hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), leads to an inhibition ofNADtdependent deacetylase sirtuin 1 (SIRT1). It then relays to theacetylation-dependent inactivation of PGC1α consequently resulting in adepressed mitochondrial biogenesis that exaggerates the NAD⁺availability. The low activity of PGC1α yields downregulated theexpressions not only of mitochondrial proteins encoded in nucleus butalso of the mitochondrial transcription factor TFAM neighboring themitochondrial DNA.

In addition to the two core senescence-regulating pathways including p53and p16/Rb, senescence-associated secretory phenotype (SASP), where anarray of inflammatory cytokines, chemokines, and proteases such as IL-1,IL-6/VEGF, IL-8, and CXCL9/MMP are released, is one of the mostcharacterized phenomena in senescence. The transcription factor GATA4 isdegraded with the association of the autophagic adaptor p62 by selectiveautophagy under normal condition, whereas DNA damage response (DDR)kinases ATM (ataxia telangiectasia mutated) and ATR (ataxiatelangiectasia and Rad3-related) received senescence signals facilitatethe dissociation between GATA4 and p62 and stabilize GATA4, in turnactivate NF-kB through TRAF3IP2 (tumor necrosis factorreceptor-associated factor interacting protein 2) and ILIA and supportSASP. SASP is completely hampered in rho0 cells (mtDNA free cellsestablished by a forced mitophagy). Mitochondrial replacement of oocytesderived from the old in an experimental IVF surely promoted the successrate for zygote formation, development and embedding of embryo, andbearing offspring.

Impaired proteostasis (protein homeostasis) is another characteristicsin aging. The integrity of proteostasis is strictly maintained bytranslational regulation, protein folding chaperon, ubiquitin-proteasomesystem (UPS), and the autophagy-lysosome system. Because chaperonesdepend upon ATP, decrease of bioenergy with aging jeopardize thefunction to correct protein folding. Both UPS and the autophagy-lysosomesystem, including mitophagy, decline with time. The alternations ofthese three systems generate aggregates which are not recycled incytosol leading to degenerative disorders. In mitochondrial matrices,the accumulation of aberrant proteins not only actuates the system todegrade them but also offers an opportunity to recover the mitochondrialfunction communicating to nucleus termed as mitochondrial unfoldedprotein response (UPR′). All the above mentioned pathways involvemitochondria. The mitochondrial replacement in somatic cells could breakthe deleterious worsening cycle of aging, slow the senescent process,and even rejuvenate cells.

Thus, the methods provided herein offer clinically viable methods totreat heteroplasmy, and/or treat various diseases, such as diseasesassociated with senescence, by replacing endogenous dysfunctionalmitochondria, such as endogenous mitochondria with mutant mtDNA, withyoung and/or healthy mitochondria that can have either an autologous orallogeneic origin.

In some embodiments, the methods provided herein for mitochondriareplacement can be used for the treatment of mitochondrial disease ordisorder, as well as senescence, cancer, and immune system deficiencies.

5.4.2 Methods of Treating a Mitochondrial Disease or Disorder

Also provided herein, are methods of treating a subject having orsuspected of having mitochondrial disease or disorder according to anyof methods described in Section 5.2 and/or Section 5.3. In someembodiments, the methods of treating a subject having or suspected ofhaving a mitochondrial disease or disorder include generating a MirCaccording to any of the methods described in Section 5.2 and/or Section5.3, and then administering a therapeutically effective amount of themitochondria replaced recipient cell to the subject having or suspectedof having a mitochondrial disease or disorder.

Various mitochondrial diseases or disorders are known, and all arecapable of being treated using the methods provided herein. For example,the mitochondrial disease or disorder capable of being treated using themethods provided herein can be a Complex I deficiency (OMIM:252010).Complex I deficiency can be caused by a mutation in any of the subunitsthereof. In another embodiment, the Complex I deficiency is caused by amutation in a gene selected from the group consisting of NDUFV1(OMIM:161015), NDUFV2 (OMIM:600532), NDUFS1 (OMIM:157655), NDUFS2(OMIM:602985), NDUFS3 (OMIM:603846), NDUFS4 (OMIM:602694), NDUFS6(OMIM:603848), NDUFS7 (OMIM:601825), NDUFS8 (OMIM:602141), and NDUFA2(OMIM:602137).

In addition, the mitochondrial disease or disorder capable of beingtreated using the methods provided herein can be a Complex IV deficiency(cytochrome c oxidase; OMIM:220110). Complex IV deficiency can be causedby a mutation in any of the subunits thereof. In certain circumstancesthe Complex IV deficiency is caused by a mutation in a gene selectedfrom the group consisting of MTCO1 (OMIM:516030), MTCO2 (OMIM:516040),MTCO3 (OMIM:516050), COX10 (OMIM:602125), COX6B1 (OMIM:124089), SCO1(OMIM:603644), FASTKD2 (OMIM:612322), and SCO2 (OMIM:604272).

Mitochondrial diseases or disorders can be caused by or associated witha mutation. The mutation can be a point mutation, a missense mutation, adeletion, and an insertion. It is understood that the identification ofmutations in mtDNA or nDNA is within the skill of those in the art, andexemplary methods are provided herein, such as, for example, a singlenucleotide polymorphism (SNP) assay or a droplet digital PCR.

Non-limiting examples of specific types of mitochondrial diseases ordisorders capable of being treated using the methods provided hereininclude Ornithine Transcarbamylase deficiency (hyperammonemia) (OTCD),Carnitine 0-palmitoyltransferase II deficiency (CPT2), Fumarasedeficiency, Cytochrome c oxidase deficiency associated with Leighsyndrome, Maple Syrup Urine Disease (MSUD), Medium-Chain Acyl-CoADehydrogenase deficiency (MCAD), Acyl-CoA Dehydrogenase Very Long-Chaindeficiency (LCAD), Trifunctional Protein deficiency, ProgressiveExternal Ophthalmoplegia with Mitochondrial DNA Deletions (POLG), DGUOK,TK2, Pyruvate Decarboxylase deficiency, and Leigh Syndrome (LS). Inanother embodiment, the mitochondrial disease or disorder is selectedfrom the group consisting of Alpers Disease; Barth syndrome;(3-oxidation defects; carnitine-acyl-camitine deficiency; carnitinedeficiency; co-enzyme Q10 deficiency; Complex II deficiency(OMIM:252011), Complex III deficiency (OMIM:124000), Complex Vdeficiency (OMIM:604273), LHON-Leber Hereditary Optic Neuropathy;MM-Mitochondrial Myopathy; LIMM-Lethal Infantile Mitochondrial Myopathy;MMC-Maternal Myopathy and Cardiomyopathy; NARP-Neurogenic muscleweakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease; FICP-FatalInfantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy;MELAS-Mitochondrial Encephalomyopathy with Lactic Acidosis andStrokelike episodes; LDYT-Leber's hereditary optic neuropathy andDystonia; MERRF-Myoclonic Epilepsy and Ragged Red Muscle Fibers;MHCM-Maternally inherited Hypertrophic CardioMyopathy; CPEO-ChronicProgressive External Ophthalmoplegia; KSS-Kearns Sayre Syndrome;DM-Diabetes Mellitus; DMDF Diabetes Mellitus+Deafness; CIPO-ChronicIntestinal Pseudoobstruction with myopathy and Ophthalmoplegia;DEAF-Maternally inherited DEAFness; PEM-Progressive encephalopathy;SNHL-SensoriNeural Hearing Loss; Encephalomyopathy; Mitochondrialcytopathy; DEMCHO-Dementia and Chorea; AMDF-Ataxia, Myoclonus; ESOCEpilepsy; Optic atrophy; FBSN Familial Bilateral Striatal Necrosis; FSGSFocal Segmental Glomerulosclerosis; LIMM Lethal Infantile MitochondrialMyopathy; MDM Myopathy and Diabetes Mellitus; MEPR Myoclonic Epilepsyand Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCMMaternally Inherited Hypertrophic CardioMyopathy; MICM MaternallyInherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome;Mitochondrial Encephalocardiomyopathy; Multisystem MitochondrialDisorder (myopathy, encephalopathy, blindness, hearing loss, peripheralneuropathy); NAION Nonarteritic Anterior Ischemic Optic Neuropathy; PEMProgressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT RettSyndrome; SIDS Sudden Infant Death Syndrome; and MIDD MaternallyInherited Diabetes and Deafness.

The methods provided herein for treating a mitochondrial disease ordisorder can also include, in specific embodiments, a mitochondrialdisease or disorder caused by mitochondrial DNA abnormalities, where themitochondrial DNA abnormalities are selected from the group consistingof chronic progressive external ophthalmoplegia (CPEO), Pearsonsyndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD),leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia,and retinitis pigmentosa syndrome (NARP), maternally-inherited Leighsyndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, andstroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiberdisease (MERRF), familial bilateral striatal necrosis/striatonigraldegeneration (FBSN), Luft disease, aminoglycoside-induced Deafness(AID), and multiple deletions of mitochondrial DNA syndrome.

Mutations in mtDNA are thought to be associated with numerous clinicaldisorders. In adults, these include neurological diseases (e.g.,migraine, strokes, epilepsy, dementia, myopathy, peripheral neuropathy,diplopia, ataxia, speech disturbances, and sensorineural deafness),gastrointestinal diseases (e.g., constipation, irritable bowel, anddysphagia), cardiac diseases (e.g., heart failure, heart block, andcardiomyopathy), respiratory diseases (e.g., respiratory failure,nocturnal hypoventilation, recurrent aspiration, and pneumonia),endocrine diseases (e.g., diabetes, thyroid disease, parathyroiddisease, and ovarian failure), ophthalmological diseases (e.g., opticatrophy, cataract, ophthalmoplegia, and ptosis). In children, disordersthought to be associated with mtDNA mutations include neurologicaldiseases (e.g., epilepsy, myopathy, psychomotor retardation, ataxia,spasticity, dystonia, and sensorineural deafness), gastrointestinaldiseases (e.g., vomiting, failure to thrive, and dysphagia), cardiacdiseases (e.g., biventricular hypertrophic cardiomyopathy and rhythmabnormalities), respiratory diseases (e.g., central hypoventilation andapnea), hematological diseases (e.g., anemia and pancytopenia), renaldiseases (e.g., renal tubular defects), liver diseases (e.g., hepaticfailure), endocrine diseases (e.g., diabetes and adrenal failure), andophthalmological diseases (e.g., optic atrophy). Accordingly, themethods and compositions provided herein are contemplated for use intreating or preventing diseases and disorders associated with mutationsin mtDNA.

In other specific embodiments, the methods provided herein allow fortreating a mitochondrial disease or disorder where the mitochondrialdisease or disorder is caused by nuclear DNA abnormalities, and thenuclear DNA abnormalities are selected from the group consisting ofMitochondrial DNA depletion syndrome-4A, mitochondrial recessive ataxiasyndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy(MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerasegamma (POLG)-related disorders, sensory ataxia neuropathy dysarthriaophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinalcord involvement and lactate elevation (LBSL), co-enzyme Q10 deficiency,Leigh syndrome, mitochondrial complex abnormalities, fumarasedeficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency,succinyl-CoA ligase deficiency, pyruvate dehydrogenase complexdeficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitinepalmitoyltransferase I (CPT I) deficiency, carnitinepalmitoyltransferase II (CPT II) deficiency, carnitine-acyl-carnitine(CACT) deficiency, autosomal dominant-/autosomal recessive-progressiveexternal ophthalmoplegia (ad-/ar-PEO), infantile onset spinal cerebellaratrophy (IOSCA), mitochondrial myopathy (MM) spinal muscular atrophy(SMA), growth retardation, aminoaciduria, cholestasis, iron overload,early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

Many individuals with a mutation of mtDNA display a cluster of clinicalfeatures that fall into a discrete clinical syndrome, such as theKearns-Sayre syndrome (KSS), chronic progressive externalophthalmoplegia (CPEO), mitochondrial encephalomyopathy with lacticacidosis and stroke-like episodes (MELAS), myoclonic epilepsy withragged-red fibers (MERRF), neurogenic weakness with ataxia and retinitispigmentosa (NARP), or Leigh syndrome (LS). However, considerableclinical variability exists and many individuals do not fit neatly intoone particular category, which is well-illustrated by the overlappingspectrum of disease phenotypes (including mitochondrial recessive ataxiasyndrome (MIRAS) resulting from mutation of the nuclear gene POLG, whichhas emerged as a major cause of mitochondrial disease or disorder.

Exemplary diseases where mitochondrial impairment is known to play animportant role include, but are not limited to, the pathogenesis of manyneurodegenerative diseases, including Alzheimer's disease, Parkinson'sdisease, Huntington's disease, and amyotrophic lateral sclerosis. Inaddition, mitochondrial disease or disorders are subtyped into a numberof syndromes according to the symptoms rather than the types ofmutations. For example, mitochondrial syndromes include Mitochondrialmyopathy, Encephalomyopathy, Lactic acidosis, Stroke-like symptoms(MELAS), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), and Leighsyndrome.

5.4.3 Methods of Treating a Subject in Need of Mitochondrial Replacement

Also provided herein, are methods of treating a subject in need ofmitochondrial replacement according to any of methods described inSection 5.2 and/or Section 5.3. In some embodiments, the methods oftreating a subject in need of mitochondrial replacement includegenerating a MirC according to any of the methods described in Section5.2 and/or Section 5.3, and then administering a therapeuticallyeffective amount of the mitochondria replaced recipient cell to thesubject in need of mitochondrial replacement.

A subject in need of mitochondrial replacement includes any subject thathas a dysfunctional mitochondria. In certain embodiments, the subject inneed of mitochondrial replacement has an age-related disease, amitochondrial disease or disorder, a neurodegenerative disease, aretinal disease, diabetes, a hearing disorder, a genetic disease, or acombination thereof. Neurodegenerative diseases that can benefit frommitochondrial replacement include, but are not limited to, amyotrophiclateral sclerosis (ALS), Huntington's disease, Alzheimer's disease,Parkinson's disease, Friedreich's ataxia, Charcot Marie Tooth diseaseand leukodystrophy. The retinal disease can be wet or dry age-relatedmacular degeneration, macular edema, or glaucoma. Other exemplarydiseases, such as age-related diseases, and/or mitochondrial disease ordisorder are described in more detail in Section 5.4.1 and 5.4.2.

A subject in need of mitochondrial replacement can also include asubject that is predisposed to mitochondrial dysfunction, and isasymptomatic. For example, the subject may have mutant mtDNA, but bewithout manifestations of, for example, a mitochondrial disease becausethe disease is an adult-onset disease. Therefore, the methods providedherein can be used to also prevent any of the diseases described hereinby treating a subject in need of mitochondrial replacement.

5.5 Methods to Produce an iPSC

The current invention also provides methods, as described in Section 5.2and Section 5.3, for producing or enhancing the production of an inducedpluripotent stem cell (iPSC) from a non-pluripotent cell. iPSCs havebeen demonstrated to be produced from non-pluripotent cells usingexogenous expression of stemness factors, such as Oct3/4, Klf4, Sox2,and c-Myc. In addition, low amount of mitochondrial DNA (mtDNA) copieshave been detected in undifferentiated ESCs, while this number increasesupon differentiation together with the level of mitochondrial maturation(Facucho-Oliveira J M, et al, J Cell Sci 2007; 120(Pt 22):4025-4034).Thus, the present invention has also identified that the methodsprovided herein can be used to enhance the generation of iPSC byreducing endogenous mtDNA in a non-pluripotent by contacting a recipientnon-pluripotent cell with an agent that reduces endogenous mtDNA,incubating the recipient non-pluripotent cell for a sufficient period oftime for the agent to partially reduce the endogenous mtDNA in thenon-pluripotent cell, and then introducing one or more expressioncassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc. In certainembodiments, exogenous mtDNA and/or exogenous mitochondria isnon-invasively transferred into the recipient cells.

It is understood that the introduction of the one or more expressioncassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc can occurprior to, during, or after introduction of the agent that reducesendogenous mtDNA. Accordingly, in some embodiments, the methods forproducing an iPSC includes introducing one or more expression cassettesfor expression of Oct3/4, Klf4, Sox2, and c-Myc, contacting a recipientnon-pluripotent cell with an agent that reduces endogenous mtDNA, andincubating the recipient non-pluripotent cell for a sufficient period oftime for the agent to partially reduce the endogenous mtDNA in therecipient cell.

In certain embodiments, the method further comprises incubating therecipient cell with an exogenous mitochondria and/or exogenous mtDNA fora sufficient period of time to non-invasively transfer the exogenousmitochondria and/or exogenous mtDNA into a recipient cell. In specificembodiments, the method further comprises incubating the recipient cellwith an exogenous mitochondria and/or exogenous mtDNA for a sufficientperiod of time to replace a majority of the endogenous mtDNA. Themethods of producing an iPSC from a non-pluripotent cell that includetransferring exogenous mitochondria and/or exogenous mtDNA and/orexogenous mitochondria can also include any of the embodiments describedin Section 5.3.

Because low amounts of mitochondrial DNA (mtDNA) copies have beendetected in undifferentiated embryonic stem cells (ESCs), the methodsprovided herein can also be used to promote pluripotency innon-pluripotent stem cells, and reduce the number of exogenous genesthat are required to generate an iPSC. For example, in some embodiments,the methods provided herein can be used to generate an iPSC by reducingendogenous mtDNA in a non-pluripotent by contacting a recipientnon-pluripotent cell with an agent that reduces endogenous mtDNA,incubating the recipient non-pluripotent cell for a sufficient period oftime for the agent to partially reduce the endogenous mtDNA in thenon-pluripotent cell, and then introducing one or more of Oct3/4, Klf4,Sox2, and c-Myc into the non-pluripotent cell, thereby generating apluripotent stem cell. In some embodiments, the iPSC can even begenerated using only small molecule agents and no exogenous factors.

In certain embodiments, the iPSC contains mutant mtDNA. For example, themutant mtDNA can contain a point mutation, such as, for example, a pointmutation in tRNA (e.g., MELAS). The mutant mtDNA can also include mtDNAwith a long deletion of mtDNA. In other embodiments, the non-pluripotentcell for use in producing iPSC is heteroplasmic. The incorporation ofmutant mtDNA can facilitate, for example, generation of disease models.

In some embodiments, the non-pluripotent recipient cells are somaticcells. In specific embodiments, the non-pluripotent cells arefibroblasts.

Culture conditions, identification, and establishment of iPSCs is withinthe skill of those in the art. For example, methods include thoseprovided in U.S. Pat. Nos. 8,058,065, and 8,278,104, which are herebyincorporated by reference in their entireties.

5.6 Assays for Measuring Heteroplasmy

As disclosed previously, mutant mtDNA and/or heteroplasmy can result indysfunctional mitochondria. Therefore, assays useful for assessingmitochondrial function and/or mtDNA mutations in connection with themethods provided herein for mtDNA replacement include any assays knownto a person skilled in the art that can be used to determine or predictthe functionality of mitochondria and/or mtDNA mutations.

By way of example, assays to determine mitochondrial function include,for example, measurement of any one of the following: secretory factorsassociated with senescence (e.g., pro-inflammatory cytokines, proteases,and growth and angiogenesis factors, such as IL-1, IL-6/VEGF, IL-8, andCXCL9/MMP); mitochondria function by using Oroboros; Mitophagy by usingKeima-Red; mitochondrial permeability; mitochondrial membrane potential;cytochrome c levels; reactive oxygen species; cell respiration;transcriptomics and proteomics for measurement of activated innateimmunity, rescission of hyperactivated glycolysis, mitigation of ERstress, repression of mTOR-S6 pathway, and activation of cell cycle;mitochondria dynamics, such as fission and fusion, observed by superfinemicroscopy, and quantified by a specialized software; or any assay knownin the art that measures mitochondrial function

Various sequencing methods can be used in combination of any of themethods provided herein to (1) detect mutant mtDNA, (2) quantifyheteroplasmy, and/or (3) evaluate or confirm transfer of exogenousmitochondria and/or exogenous mtDNA. A stretch of roughly 1,100nucleotides is gene-free that been called D-Loop, Displacement Loop, andControl Region. The D-Loop contains two regions within which mutationsaccumulate more frequently than anywhere else in the mitochondrialgenome. The regions are called hypervariable regions HV1 and HV2,respectively. Accordingly, in some embodiments, mtDNA mutations can beidentified in connection with the methods provided herein, by sequencingthe hypervariable regions (HV) (i.e., HV1 and/or HV2) of the D-loop ofmtDNA. mtDNA sequencing can be performed using any sequencing methodknown in the art. In specific embodiments, the sequencing methodcomprises a single nucleotide polymorphism (SNP) assay. In otherembodiments, the sequencing method comprises digital PCR. In specificembodiments, the digital PCR is droplet digital PCR.

5.7 Compositions

Also provided herein are compositions of cells obtained by any of themethods described in Sections 5.2-5.5. In certain embodiments, providedherein is a composition comprising one or more mitochondria replacedcells obtained by the method of (a) contacting a recipient cell with anagent that reduces endogenous mtDNA copy number; (b) incubating therecipient cell for a sufficient period of time for the agent topartially reduce the endogenous mtDNA copy number in the recipient cell;and (c) co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA has been partially reduced, and (2) exogenousmitochondria for a sufficient period of time to non-invasively transferthe exogenous mitochondria into the recipient cell, thereby generating amitochondria replaced cell, wherein said mitochondria replaced cellcomprises greater than 5% of exogenous mtDNA. In other aspects, providedherein is a composition comprising one or more mitochondria replacedcells obtained by the method of (a) contacting a recipient cell with anagent that reduces endogenous mtDNA copy number; (b) incubating therecipient cell for a sufficient period of time for the agent topartially reduce the endogenous mtDNA copy number in the recipient cell;and (c) co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA has been partially reduced, and (2) exogenous mtDNAfrom healthy donor, for a sufficient period of time to non-invasivelytransfer exogenous mtDNA into the recipient cell, thereby generating amitochondria replaced cell, thereby generating a mitochondria replacedcell, wherein said mitochondria replaced cell comprises greater than 5%of exogenous mtDNA.

The compositions can also be obtained by a method that involvescontacting a cell with an agent that reduces mitochondrial function, andthen incubating the recipient cell for a sufficient period of time forthe agent to partially reduce endogenous mitochondrial function in therecipient cell. In some embodiments, the recipient cell having partiallyreduced endogenous mitochondrial function can then be co-incubated witheither exogenous mitochondria from a healthy donor, for a sufficientperiod of time to non-invasively transfer exogenous mitochondria intothe recipient cell, thereby generating a mitochondria replaced cell. Inother embodiments, the recipient cell having partially reducedendogenous mitochondrial function can then be co-incubated with eitherexogenous mtDNA from a healthy donor, for a sufficient period of time tonon-invasively transfer exogenous mitochondria into the recipient cell,thereby generating a mitochondria replaced cell. In some embodiments,the mitochondria replaced cell generated by the methods described abovecomprises greater than 5% of exogenous mtDNA.

As described above, the exogenous mitochondria can be comprised ofexogenous mtDNA. Therefore, in some embodiments both exogenousmitochondria and exogenous mtDNA are transferred to the recipient celland the MirC has both exogenous mitochondria and exogenous mtDNA. Inother embodiments, the exogenous mtDNA is transferred to the recipientcell via exogenous mitochondria, and then the exogenous mtDNA isdelivered to the endogenous mitochondria. Under certain circumstancesthe exogenous mitochondria is removed from the cell after the exogenousmtDNA is delivered to the endogenous mitochondria. Accordingly, in someembodiments, the MirC have exogenous mtDNA and does not have exogenousmitochondria.

Because the endogenous mtDNA of the recipient cell is partiallydegraded, the MirC that includes exogenous mitochondria, exogenousmtDNA, or a combination thereof can contain both exogenous mtDNA andendogenous mtDNA. Similarly, in scenarios where the exogenousmitochondria is transferred to the recipient cell, the MirC can containboth exogenous mitochondria and endogenous mitochondria. Thus, inspecific embodiments, the compositions of one or more mitochondriareplaced cells obtained by the methods provided herein have a mixture ofendogenous and exogenous mitochondria. In other embodiments, thecompositions of one or more mitochondria replaced cells obtained by themethods provided herein have a mixture of endogenous mtDNA and exogenousmtDNA (i.e., heteroplasmic mtDNA). In yet further embodiments, the oneor more mitochondria replaced cells have a total mtDNA copy number thatis no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copynumber of the recipient cell prior to contacting with the agent thatreduces endogenous mtDNA copy number.

The present invention also includes compositions for use in a method ofgenerating mitochondria replaced that includes an agent that reducesendogenous mtDNA or an agent that reduces mitochondrial function, and asecond active agent. In certain embodiments, the composition can furtherinclude an exogenous mitochondria, one or more recipient cells, or acombination thereof. In yet further embodiments, the composition canfurther include exogenous mtDNA.

As described in Section 5.3, various second active agents can be used inthe methods for generating one or more mitochondria replaced cells. Forexample, in some embodiments, the second active agent includes largemolecules, small molecules, or cell therapies, and the second activeagent is optionally selected from rapamycin, NR (Nicotinamide Riboside),bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide(MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thiocticacid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis.

The use of an activator of endocytosis was found to enhance the uptakeof exogenous mitochondria in cells treated with the MTS-XbaIR plasmid,but had no effect on promoting uptake with “add on” or mock transfectedcells, which indicated that this mechanism of transferring exogenousmitochondria was unique to the invention provided herein. Non-limitingexemplary compounds suitable for activating endocytosis include, forexample, phorbol-12-myristate-13-acetate (PMA) (C₃₆H₅₆O₈),12-O-tetradecanoylphorbol 13-acetate (TPA) (C₃₆H₅₆O₈), tanshinone IIAsodium sulfonate (TSN-SS) (C₁₉H₁₇O₆S.Na), and phorbol-12,13-dibutyrate,or derivatives thereof. In some embodiments, the activator ofendocytosis comprises a modulator of cellular metabolism.

Modulating cellular metabolism may be accomplished by any of a number ofwell-known techniques including but not limited to those describedherein, and in the cited references. For example, in some embodiments,modulating cellular metabolism is performed by nutrient starvation ornutrient deprivation. In other embodiments, modulating cellularmetabolism is performed by a chemical inhibitor or small molecule. Inspecific embodiments, the chemical inhibitor or small molecule is anmTOR inhibitor.

Various compounds are known to inhibit mTOR, including rapamycin, alsoknown as sirolimus (CAS Number 53123-88-9; C₅₁H₇₉NO₁₃), and rapamycinderivatives (e.g., rapamycin analogs, also known as “rapalogs”).Rapamycin derivatives include, for example, temsirolimus (CAS Number162635-04-3; C₅₆H₈₇NO₁₆), everolimus (CAS Number 159351-69-6;C₅₃H₈₃NO₁₄), and ridaforolimus (CAS Number 572924-54-0; C₅₃H₈₄NO₁₄P).Accordingly, in some embodiments, the compositions provided hereincomprise rapamycin or a derivative thereof. It is understood that theembodiments described above for modulating cellular metabolism arenon-limiting, and modulating cellular metabolism need not involve achemical compound or small molecule, and can include modulation of otherpathways beyond mTOR. It is also understood that the compositions canoptionally comprise activators of endocytosis, and that it is not arequired component. In addition, in some embodiments the inventionprovided herein can involve non-endocytosis mediated transfer of mtDNAand/or mitochondria, such as in non-clinical settings.

As described in Section 5.5, the present invention also provides, incertain embodiments, a composition for use in a method of producing aninduced pluripotent stem cells (iPSC) from a non-pluripotent cell thatincludes an agent that reduces endogenous mtDNA, one or more expressioncassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc, and arecipient cell, wherein the recipient cell is a non-pluripotent cell,wherein the agent that reduces endogenous mtDNA is present in an amounteffective to increase efficiency of producing an induced pluripotentstem cells (iPSC) from a non-pluripotent cell, as compared to anon-pluripotent cell not treated with an agent that reduces endogenousmtDNA. In some embodiments, the agent that reduces endogenous mtDNA ispresent in an amount effective to increase efficiency of producing aninduced pluripotent stem cells (iPSC) from a non-pluripotent cell, ascompared to a non-pluripotent cell not treated with an agent thatreduces endogenous mtDNA. This is based in part on the observation thatpluripotent cells have a reduction in mtDNA copy number. In specificembodiments, the composition for use in a method of producing an iPSCfurther comprises exogenous mitochondria and/or exogenous mtDNA.

The present invention also includes pharmaceutical compositions for usein the treatment of an age-related disease, a mitochondrial disease ordisorder, a neurodegenerative disease, diabetes, a genetic disease, orany subject in need of mitochondrial replacement, as described inSection 5.4. In certain embodiments, provided herein are pharmaceuticalcompositions that include an isolated population of mitochondriareplaced cells that have exogenous mitochondria from a healthy donor,and the cells are obtained by the methods described herein, such as inSections 5.2-5.3. In other embodiments, the pharmaceutical compositionincludes an isolated population of mitochondria replaced cells withexogenous mitochondria and/or exogenous mtDNA from a healthy donor, andthe cells are obtained by the methods described herein, such as inSections 5.2-5.3. For example, in some embodiments, the mitochondriareplaced cells that have exogenous mtDNA can optionally further includeexogenous mitochondria. In other embodiments, the exogenous mtDNA istransferred into the cell via exogenous mitochondria, delivered to theendogenous mitochondria, and then the exogenous mitochondria is removedfrom the recipient cell.

The disclosure also provides a pharmaceutical composition comprising anisolated population of mitochondria replaced cells having an exogenousmitochondria from a healthy donor, wherein the cells are obtained by anyof the methods provided herein for obtaining a mitochondrial replacedcell. In yet another aspect, the disclosure provides a pharmaceuticalcomposition comprising an isolated population of mitochondria replacedcells having an exogenous mtDNA from a healthy donor, wherein the cellsare obtained by any of the methods provided herein for obtaining amitochondrial replaced cell. In some embodiments, the pharmaceuticalcomposition comprising an isolated population of mitochondria replacedcells having an exogenous mtDNA from a healthy donor further comprisesexogenous mitochondria.

For example, in some embodiments, a pharmaceutical compositioncomprising an exogenous mitochondria from a healthy donor are obtainedby a method that involves contacting a cell with an agent that reducesmtDNA copy number, and then incubating the recipient cell for asufficient period of time for the agent to partially reduce endogenousmtDNA copy number in the recipient cell. In some embodiments, therecipient cell having partially reduced endogenous mtDNA copy number canthen be co-incubated with either exogenous mitochondria from a healthydonor, for a sufficient period of time to non-invasively transferexogenous mitochondria into the recipient cell, thereby generating amitochondria replaced cell. In other embodiments, the recipient cellhaving partially reduced endogenous mtDNA copy number can then beco-incubated with either exogenous mtDNA from a healthy donor, for asufficient period of time to non-invasively transfer exogenousmitochondria into the recipient cell, thereby generating a mitochondriareplaced cell. In some embodiments, the mitochondria replaced cellgenerated by the methods described above comprises greater than 5% ofexogenous mtDNA.

In other embodiments, the cells are obtained by a method that involvescontacting a cell with an agent that reduces mitochondrial function, andthen incubating the recipient cell for a sufficient period of time forthe agent to partially reduce endogenous mitochondrial function in therecipient cell. In some embodiments, the recipient cell having partiallyreduced endogenous mitochondrial function can then be co-incubated witheither exogenous mitochondria from a healthy donor, for a sufficientperiod of time to non-invasively transfer exogenous mitochondria intothe recipient cell, thereby generating a mitochondria replaced cell. Inother embodiments, the recipient cell having partially reducedendogenous mitochondrial function can then be co-incubated with eitherexogenous mtDNA from a healthy donor, for a sufficient period of time tonon-invasively transfer exogenous mitochondria into the recipient cell,thereby generating a mitochondria replaced cell. In some embodiments,the mitochondria replaced cell generated by the methods described abovecomprises greater than 5% of exogenous mtDNA. The agent that reducesmitochondrial function can either transiently or permanently reducemitochondrial function. It is within the skillset of a person skilled inthe art to be able to determine whether the agent would transiently(e.g., reversible inhibitor) or permanently (e.g., irreversibleinhibitor) reduces mitochondrial function.

In certain embodiments of the pharmaceutical compositions providedherein, the cells are obtained by a method further comprising furthercomprising contacting the recipient cell with a second active agentprior to co-incubating the recipient cell with exogenous mitochondriaand/or exogenous mtDNA. In some embodiments, the second active agent isselected from the group consisting of large molecules, small molecules,or cell therapies, and the second active agent is optionally selectedfrom the group consisting of rapamycin, NR (Nicotinamide Riboside),bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide(MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thiocticacid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis. In specific embodiments, the activator ofendocytosis is a modulator of cellular metabolism. In other embodiments,the modulator of cellular metabolism comprises nutrient starvation, achemical inhibitor, or a small molecule. In further embodiments, thechemical inhibitor or the small molecule is an mTOR inhibitor. In yetfurther embodiments, said mTOR inhibitor comprises rapamycin or aderivative thereof.

As described above in Section 5.2, various types of cells can be used asrecipient cells and donor cells. For example, the present disclosuredescribes numerous examples where the recipient cells are mammaliancells. However, it is also understood that any cell with a mitochondriacan be a recipient cell. Therefore, the recipient cell can also be aplant cell.

In some embodiments, the animal cells are mammalian cells. In specificembodiments, the cells are somatic cells. In further embodiments, thesomatic cells are epithelial cells. In yet further embodiments, theepithelial cells are thymic epithelial cells (TECs).

The present disclosure also provides compositions where the somaticcells are immune cells. For example, the compositions can compriseimmune cells where the immune cells are T cells, such as exhausted Tcells. In some embodiments, the composition includes rejuvenated T cellsthat contain exogenous mitochondria and/or exogenous mtDNA. For example,senescent T cells or near senescent T cells (e.g., immunosenescent) canserve as a recipient cell and a T cell-derived MirC can be generatedusing the methods provided herein to produce a T cell with healthyexogenous mitochondria and/or exogenous mtDNA. In specific embodiments,the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+T cells. In some embodiments, the T cells are chimeric antigen receptor(CAR) T cells. For example, in some embodiments the disclosure providesa MirC that is CAR-T cell, which is efficacious in killing a cancercell. The MirC derived CART can have prolonged survival to enableincreased immunosurveillance, and enhanced cancer cell killing. In otherembodiments, the immune cells are phagocytic cells.

As described above, the compositions provided herein can also include acomposition for use in delaying senescence and/or extending lifespan ina cell. The composition can include a senescent or near senescent cellhaving endogenous mitochondria, isolated exogenous mitochondria from anon-senescent cell, and an agent that reduces endogenous mtDNA copynumber. The composition can also include a senescent or near senescentcell having endogenous mitochondria, isolated exogenous mitochondriafrom a non-senescent cell, and an agent that reduces mitochondrialfunction.

Also provided herein are compositions that include one or moremitochondria replaced cells that are derived from recipient cells thatare bone marrow cells. In specific embodiments, the bone marrow cellsare a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC).For example, an HSC or MSC can be isolated from a subject having orsuspected of having a mitochondrial disease, an age-related disease, orotherwise be in need of mitochondrial replacement, and have theendogenous mitochondria replaced with exogenous mitochondria.Subsequently, the HSC or MSC derived MirC can then be transplanted backinto the subject in need of mitochondrial replacement. In yet furtherembodiments, the recipient cells are iPS cells. The compositions can beused in the clinical setting and can be efficacious in treating anage-related disease, treating a mitochondrial disease or disorder,treating a neurodegenerative disease, treating diabetes, or a geneticdisease. For example, in some embodiments, the iPSC can bedifferentiated into a particular cell type prior to administering backinto the subject, using methods known in the art.

In other embodiments, provided herein are pharmaceutical compositionsthat include an isolated population of pluripotent cells having areduced amount of endogenous mtDNA, wherein the cells are obtained byany of the embodiments described in Section 5.5. In specificembodiments, the isolated population of pluripotent cells are iPS cells.

Administration of cells or compounds described herein is by any of theroutes normally used for introducing pharmaceuticals. The pharmaceuticalcompositions of the invention may comprise a pharmaceutically acceptablecarrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized foreign pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Pharmaceutically acceptable carriers are determined inpart by the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985).

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally, nasally, topically, intravenously,intraperitoneally, intrathecally or into the eye (e.g., by eye drop orinjection). The formulations of compounds can be presented in unit doseor multi-dose sealed containers, such as ampoules and vials. Solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

The dose administered to a patient, in the context of the presentinvention should be sufficient to induce a beneficial response in thesubject over time, i.e., to prevent, ameliorate, or improve a conditionof the subject. The optimal dose level for any patient will depend on avariety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, and on a possible combination with other drug. The size of thedose also will be determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularcompound or vector in a particular subject. Administration can beaccomplished via single or divided doses.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

All patents, applications, published applications, and otherpublications cited herein are incorporated herein by reference in theirentirety. In the event that any description of terms set forth conflictswith any document incorporated herein by reference, the description ofterm set forth herein shall control.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

EXAMPLES

The examples in this section are offered by way of illustration, and notby way of limitation. The following examples are presented as exemplaryembodiments of the invention. They should not be construed as limitingthe broad scope of the invention.

Example I: Optimization of the MirC Protocol Revealed that XbaI DegradedmtDNA In Vitro and the MTS Expression Vector Targeted Mitochondria

A scheme of the method used to generate a mitochondria replaced cell(MirC) is provided in FIG. 1A. First, the mammalian expression vectorused to express the XbaI restriction enzyme fused to amitochondrial-targeted sequence (MTS) was engineered by cloning theMTS-XbaI sequence into the pCAGGS vector using standard techniques knownin the art (FIG. 1B). Among mitochondrial transfer signals (MTS) beingreported we utilized the ND4 signal sequence in this study. Theresultant expression vector also contained the puromycin resistance geneto allow for selection (FIG. 1B).

XbaIR is one of the most powerful endonucleases and a standard sequenceof mtDNA named under Cambridge reference sequence (CRS) in humanmitochondria genome has as many as five recognition sites targeted bythe particular endonuclease (FIG. 1D). It was verified by an in vitroendonuclease co-incubation that isolated mtDNA was digested at multiplesites by XbaIR (FIG. 1C). In contrast, NotI digestion of mtDNA showed asingle fragment, as predicted by Cambridge Reference Sequence (CRS) ofmitochondrial DNA (FIG. 1C).

The gene transfer protocol of plasmid DNA to cells was optimized usingNormal Human Dermal Fibroblast (NHDF) cells that expressed enhancedgreen fluorescent protein (EGFP) by using the Nucleofectorelectroporation-based transfection method. Following one day of 2 μg/mlpuromycin exposure, an efficacy of more than 90% and viability of morethan 90% was established (FIG. 1E).

To specifically evaluate the effectiveness of the MTS targetingsequence, a plasmid carrying MTS fused with EGFP was generated bysubcloning the EGFP gene in place of the XbaIR gene to generate thepCAGGS-MTS-EGFP-PuroR plasmid (FIG. 1F). Then normal human dermalfibroblasts (NHDF) were transfected with the MTS-EGFP expression vectorand the cells were counter stained with TMRM (tetramethylrhodamine,methyl ester), which is a cell-permeant dye that accumulates in activemitochondria with intact membrane potential (FIG. 1G).

Taken together, these results demonstrated that XbaI could be used todigest mitochondrial DNA, the cells could be efficiently transfectedwithout effecting cell viability, and the expression vectors containinga MTS could effectively target mitochondria.

Example II: Endonuclease MTS-XbaIR Treatment Exhibits ImprovedDegradation of mtDNA Relative to the Conventional Method of EtBr

The efficiency and efficacy of the MTS-XbaIR expression vector relativeto the conventional method that employs ethidium bromide (EtBr) wasevaluated according to the scheme illustrated in FIG. 2A. The placentalvenous endothelium-derived cell line EPC100 with DsRed labeledmitochondria were cultured in pyruvate-free DMEM (Wako cat #044-29765)with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S),and at day 0 the cells were either untreated (“normal”), transfectedwith the MTS-XbaIR expression vector (“MTS-XbaIR”), or treated with 50ng/mL of EtBr. At day 1, the cells were cultured in DMEM with 10% FBS,and 1% P/S supplemented with 100 μg/mL pyruvate and 50 μg/mL uridine.Quantitative polymerase chain reaction (qPCR) was performed according tomethods known in the art at days 3 and 5 to measure the mtDNA relativeto the housekeeping gene, (3-actin (Actb). The results demonstrated thatXbaIR reduced the mtDNA copy number to 2715.8141, whereas the EtBrtreatment only reduced the mtDNA copy number to 5169.1258, which wassimilar to the DNA copy number of 6189.6867 in untreated cells (FIG.2B). While the reduction of mtDNA in the endonuclease treated group wassuperior to that of the group treated with the conventional method, itwas not a complete deletion and approximately 30% of the endogenousmtDNA remained (FIG. 2B). Cells with this partial reduction of mtDNAwere termed as ρ(−) cells.

The enhanced degradation of endogenous mtDNA in the MTS-XbaIR treatedgroup, relative to the EtBr group was further confirmed by microscopy ofthe DsRed labeled mitochondria (FIG. 2C). The level of reduction wasreflected in the remaining healthy mitochondrial volume estimated by theTMRM staining, which was lower in the XbaIR treated group than that withthe conventional method (FIG. 2C). In addition, a FACS analysis of NHDFcells demonstrated a reduction of TMRM after treating with XbaIR (FIG.2D).

The kinetics of the expression of XbaIR following plasmid gene transferwas examined by qPCR. On day 3, the expression reached the peak and thendeclined to zero on day 7 (FIG. 2E). Other genes of interest (e.g., GFP)verified the same kinetics as XbaIR (FIG. 2E). Fluorescent imagesconfirmed the enrichment of GFP following in cells transfected with theMTS-EGFP-PuroR plasmid after puromycin selection, as compared to beforetransfection (FIG. 2F and FIG. 2G). The fraction of GFP positive cellssignificantly increased to almost 100% by exposing the puromycin for oneday (FIG. 2F).

These results demonstrated that the reduction of mtDNA copy number inthe XbaI endonuclease treated group was superior to that of the grouptreated with the conventional method of EtBr, and did not completelydelete all of the endogenous mtDNA. Moreover, a brief selection usingpuromycin enabled significant enrichment of the cells expressing the MTSconstruct.

Example III: Partial Degradation of Endogenous Mitochondria UsingMTS-XbaIR Construct in Recipient Cell Enabled Mitochondria Replacementfrom Exogenous Donor Cell

To evaluate whether exogenous mitochondria from a healthy donor cellcould be transferred to a recipient cell with XbaI mediated depletion ofmtDNA, NHDF cells were transfected with the MTS-GFP or MTS-XbaIRplasmids and selected using puromycin after 48 hours. After 6 dayspost-transfection, isolated mitochondria from human cell linesoriginated from the endothelium of the uterus (named as EPC100) thatwere labeled with DsRed were transferred to the donor cells. A scheme ofthe protocol is shown in FIG. 3A.

Following transfection with MTS-GFP or MTS-XbaI and selection withpuromycin the mitochondria content was evaluated by TMRM staining. Asshown in FIG. 3B, the MTS-GFP transfected cells exhibited a strongstaining for TMRM, indicating high levels of mitochondria in the NHDFcells. In contrast, the MTS-XbaI transfected cells (ρ−) exhibited areduction of the mitochondrial volume, as visualized by TMRM staining(FIG. 3B).

The reduction of mitochondrial DNA was further confirmed by quantifyingthe number of mitochondrial DNA copies by qPCR of 12S-rRNA afteradjusting with β-actin (Actb) in the nucleus (FIG. 3C). On day 6, therewas a significant reduction in mitochondrial DNA from MTS-XbaItransfected cells (ρ−), relative to the NHDF control cells transfectedwith MTS-GFP (FIG. 3C). The significant reduction in mitochondrial DNAin the ρ-cells continued for the length of the assay, which was stoppedon day 12. Specifically, the copy numbers dropped to about ⅓ of theoriginal copy numbers on day 6 and further declined to about ¼ on day 12in the ρ-cells (FIG. 3C).

Mitochondria were isolated from DsRed-Mt EMCs by differentialcentrifugation. In brief, the cells were harvested from culture disheswith homogenization buffer [HB; 20 mM HEPES-KOH (pH 7.4), 220 mMmannitol and 70 mM sucrose] containing a protease inhibitor mixture(Sigma-Aldrich, St. Louis, Mo., USA). The cell pellet was resuspended inHB and incubated on ice for 5 min. The cells were ruptured by 10 strokesof a 27-gauge needle on ice. The homogenate was centrifuged (400 g, 4°C., 5 min.) two times to remove unbroken cells. The mitochondria wereharvested by centrifugation (6000 g, 4° C., 5 min.) and resuspended inHB. The amounts of isolated mitochondria were expressed as proteinconcentration using a Bio-Rad protein assay kit (Bio-Rad, Richmond,Calif., USA). Mitochondrial transfer was conducted by co-incubatingisolated mitochondria with cells in 2 ml of standard medium at 37° C.under 5% CO2 for 24 h. Importantly, the co-incubation of isolatedmitochondria with ρ(−) cells on day 12 resulted in a significantincrease in mtDNA copy number, similar to the levels of control NHDFcells (FIG. 3C).

Consistent with the results shown in FIG. 2C and FIG. 2D, the ρ(−) cellsexhibited reduced mitochondria content after MTS-XbaI transfection, asmeasured by visualization of TMRM. Importantly, the decrease inmitochondria could be rescued by contacting the ρ(−) cells with theisolated exogenous mitochondria, as indicated by the uptake of the DsRedlabeled isolated mitochondria (FIG. 3D and FIG. 3E). In contrast, theco-cultivation of DsRed-marked and isolated mitochondria with eitherNHDF control cells or NHDF cells transfected with the mock transfectantMTS-EGFP expression vector revealed that the exogenous mitochondriagathered around the cells and formed aggregates, but failed to beinternalized (FIG. 3D, lower panels). Although a minor portion of themitochondria were engulfed, most of them stayed outside of the cellswith intact endogenous mitochondria, and the intensity of DsRed wasmaintained during this period. The aggregates of DsRed became smaller,and fewer, and the intensity of DsRed was reduced suggesting that aftergathering exogenous mitochondria onto cell membrane, they were engulfedand their membranous portions of mitochondria were rapidly digested.

Comparison between existing methods demonstrated that the endonucleasemethod of the present invention was more efficacious in generating amitochondria replaced cell having exogenous mitochondria (FIG. 3F). Forexample, the endonuclease method of the present invention was comparedwith (1) the add-on mitochondria transfer method, described in ourprevious works (see, e.g., Kitani, T., et al, J Cell Mol Med (2014) 18,1694) or (2) a recently reported method (see, e.g., Kim, M. J., et al.,Sci Rep 8, 3330, (2018)) that employed spinoculation of isolatedmitochondria with metabolically healthy cells (FIG. 3F). Neither of thepreviously reported methods (i.e., mitochondria add-on; “Mt add-on”, orspinoculation at 800×g or 1500×g) demonstrated any significant transferof exogenous mitochondria, as measured by FACS analysis of DsRed labeledexogenous mitochondria (FIG. 3F). On the other hand, the novel methodprovided herein that employed MTS-XbaI mediated partial degradation ofendogenous mitochondria followed by the non-invasive transfer orexogenous mitochondria (Mt EPC100) demonstrated a significant DsRedpositive fraction and increased mean fluorescence intensity aftertransfer of exogenous mitochondria (FIG. 3F, top right graph, far rightline).

Previously established methods used in mitochondria biology utilizedcells with a complete deletion of mitochondria ρ(0) cells (see, e.g.,U.S. patent application Ser. No. 12/747,771, filed Sep. 23, 2010, andpublished as US 2011-0008778 A1, which is incorporated by referenceherein in its entirety). However, the ρ(0) cells failed to engulfexogenous mitochondria (FIG. 3G-FIG. 3I). Based on these resultspresented herein, it was hypothesized that the ρ(0) cells failed toengulf exogenous mitochondria due to a shortage of energy necessary toundergo macropinocytosis. To confirm the hypothesis, we designed genemodified cells to generate ρ(0) cells with an exposure to antimycin thatinduces mitophagy, and examined the mitochondria transfer level. Theresults demonstrated that no engulfment of exogenous mitochondriaoccurred in cells with a complete deletion of mitochondria (FIG. 3G-FIG.3I). Therefore, these results suggested that a partial deletion of thepre-existing mtDNA, rather than a complete deletion, was a key factor inthe macropinocytosis of exogenous and extracellular mitochondria.

In addition, the uptake of DsRed labeled exogenous mitochondria wasmonitored in ρ(−) cells treated with or without exogenous mitochondria,untransfected cells (add on Mt), or cells treated with the mock MTS-GFPplasmid. The fluorescent intensities of DsRed was quantified every 24hours using NIH image software. The relative values to the initialintensities were depicted in bar graph (FIG. 3J). The quantificationdemonstrated that simple add-on mitochondria coincubation andmitochondria coincubation with mock-transfectant increased theintensities at the same rate due to aggregation of the isolatedmitochondria, indicating accumulation rather than engulfment of theDs-red labeled mitochondria. In contrast, the intensity of ρ(−) cellsco-incubated with isolated, exogenous mitochondria gradually decreasedwith time, suggesting that engulfed mitochondria were degraded.

These results demonstrate that the MTS-XbaI expression vector cangenerate ρ(−) cells that have a partial deletion of endogenousmitochondria, and the mitochondrial content can be rescued bytransferring isolated exogenous mitochondria from donor cells. Asdescribed herein, the methods of the current invention provide improvedefficiency of mitochondrial transfer, relative to previously describedmethods, such as those performed in combination with centrifugation, orsimple “adding on” the mitochondria without partially reducing theendogenous mtDNA. However, mitochondrial transfer was unable to beperformed in cells with a complete degradation of endogenousmitochondria (ρ(0) cells), which indicated that the uptake of exogenousmitochondria likely requires energy.

Example IV: Isolated Exogenous Mitochondria Fuse with EndogenousMitochondria to Transfer Donor mtDNA

To further elucidate how mitochondrial transfer of intact mitochondriaoccurs, the fate of transferred mitochondria into cells was investigatedseparately on outer and inner membrane and nucleoid. In certaincircumstances, transient intermitochondrial fusion events have beenobserved, where two mitochondria came into close apposition, exchangedsoluble inter-membrane space and matrix proteins and re-separated,preserving the original morphology (see, e.g., Liu X et al., EMBO J.2009; 28(20):3074-3089; Huang X et al. Proc Natl Acad Sci US A. 2013;110(8):2846-2851). Therefore, transient intermitochondrial fusion eventswere analyzed under the conditions described herein.

Isolated mitochondria from EPC100 donor cells was labeled with DsRed,and recipient cells with EGFP-marked mitochondria were used. A diagramof the protocol employed is illustrated in FIG. 4A. Microscopy images ofthe temporal contact of the donor and resident mitochondria revealedthat no broad mitochondrial fusion was observed (FIG. 4B and FIG. 4C).The majority of the donor mitochondria existed separately from theendogenous mitochondria. In addition, a few transient fusion images wereobserved, and then the donor mitochondria appeared to run away before itfinally disappeared (FIG. 4C).

Mitochondrial transfer was performed according to the protocolillustrated in FIG. 4F. Briefly, the mitochondria of the recipient NHDFcells was marked with DsRed-marked (FIG. 4D), and mitochondria from thedonor EPC100 cells was marked with TFAM, which binds to mtDNA and allowstracing of mitochondria (FIG. 4E). The recipient NHDF cells weretransfected with the pCAGGS-MTS-XbaIR-P2A-PuroR expression vector, andselected with puromycin on day 2 for 24 hours. On day 6, mitochondrialtransfer from TFAM-GFP labeled mitochondria from EPC100 donor cells wasperformed. Then on day 8, the cells were imaged. Microscopy of themitochondrial transfer revealed that the donor nucleoid settled in thepre-existing mitochondrial matrices (FIG. 4G). The exogenousmitochondria transiently contacted the mitochondria of recipient,suggesting that mitochondrial nucleoids including TFAMs were transferredto the pre-existing mitochondria via the transient contact.

These results demonstrate that donor mitochondria were transferred intothe mitochondrial matrices in the recipient cells and dominate under thereduction of pre-existing mitochondria. Moreover, according to theseexperiments, almost all isolated mitochondria were engulfed. On theother hand, add-on type of mitochondrial transfer and mock-transfectantdid not exhibit the rigorous engulfment, but aggregated major part ofthese exogenous mitochondria onto the cellular surface.

In summary, the results from Examples III and IV demonstrate that ρ(−)cells degraded the engulfed mitochondria (FIG. 3J), and that theexogenous mitochondria temporally contacted with the pre-existingmitochondria (FIG. 4B-FIG. 4C), while the exogenous mtDNA with TFAMexisted in the pre-existing mitochondria (FIG. 4G).

Accordingly, it is hypothesized that the exogenous mitochondria are ableto briefly interact with the endogenous mitochondria, and transportmtDNA during the brief contacts. Then, the exogenous mitochondrialmembrane complexes can be degraded in the cytosol to provide buildingblocks for the reconstituted mitochondria. The mitochondria of therecipient cell that receive the exogenous mitochondria are able togradually reconstitute the mitochondrial membrane complex, anddemonstrate the functional recovery.

Example V: SNP Assay Detected Increase in Exogenous Mitochondria afterTransfer of Isolated Exogenous Mitochondria

To assess the origin of mtDNA following the mtDNA replacement, thedifferent nucleotides identified between NHDF and EPC100 by sequencingthe hypervariable region 1 and 2 were used (FIG. 5A and FIG. 5B). WhileNHDF preserves A at the position of 16362 in CRS, EPC100 harbors amutation at the same position that resulted in a change from A to G(FIG. 5B). Importantly, evaluation of the mitochondria replaced ρ(−)cells (NHDF ρ(−) Mt) demonstrated the presence of both the originalnucleotide in a minor wave and the exogenous nucleotide G in a majorwave, which indicated that the cells were heteroplasmic (FIG. 5B, bottompanel).

The heteroplasmy in the mitochondria replaced NHDF was further evaluatedby the single nucleotide polymorphism assay to detect the differencebetween the recipient NHDF and the donor EPC100 (FIG. 5C). The HV1region was amplified using the hmt16318-F primer(5′-agccatttaccgtacatagcacatt-3′ (SEQ ID NO: 6)) and the hmt16414-Rprimer (5′-cacggaggatggtggtcaag-3′ (SEQ ID NO: 9)), and the SNP wasdetected using the NHDF specific probe (5′-CTTCTCGTCCCCATG-3′ (SEQ IDNO: 5)) and the EPC100 specific probe (5′-CCCTTCTCGCCCCCAT-3′ (SEQ IDNO: 7)) (FIG. 5C). The SNP assay results demonstrated that the ratio ofEPC100 versus NHDF reached 66.6% on day 12 after the mtDNA replacement(FIG. 5D). This result was an unexpected improvement from previousmethods, which resulted in a relatively small portion of extracellularmitochondria being engulfed by human uterine endometrial gland-derivedmesenchymal cells and had little effect on the heteroplasmy levels (see,e.g., Kitani, T., et al., Journal of Cellular and Molecular Medicine,18, 1694-1703 (2014)).

These results demonstrate that the methods provided herein forreplacement of mtDNA with exogenous mitochondria and/or exogenous mtDNAis completely novel, and an improvement over the existing technology. Asdescribed herein, the methods provided demonstrate that the transfer ofmitochondria after MTS-XbaI mediated degradation of endogenousmitochondria can result in exogenous mtDNA being the predominant mtDNA.

Example VI: Replaced Mitochondria Generate Energy and MirC ExhibitPhenotypic Recovery Similar to Normal Control Cells

Whether the replaced mitochondria work to generate energy wasinvestigated by using Oroboros 02k according to the manufacturer'sinstructions. Representative oxygen consumption rate curves with nativecontrol cells, the ρ(−) cells, and the mitochondria replaced cells weregenerated and then the respiratory flow and control ratio was calculated(FIG. 6A and FIG. 6B). Basal respiration, the maximum capacity ofelectron transfer system, and ATP production (Free Routine Activity) allshowed similar kinetics and indicated that these indices significantlydropped with the ρ(−) cells (FIG. 6B, upper row). Importantly, theseindices recovered to the original values with the mitochondria replacedcells (FIG. 6A and FIG. 6B). Non-mitochondrial ATP production (ROX) wasupregulated, and the coupling ratio was downregulated in the ρ(−) cells(FIG. 6B, lower row). The energy providing machinery in the ρ(−) cellsinclined to glycolysis from mitochondrial ATP generation and the changeswere reversed after the mtDNA replacement with the native cells (FIG.6B, upper right).

In addition, the phenotypic recovery of the mitochondria replaced cells(MirC) was demonstrated by their proliferative capability (FIG. 6C).Specifically, the ρ(−) cells showed a poor proliferative capability,whereas the MirC recovered to levels near that of the control cells bydays 6-12 (FIG. 6C, right).

These results demonstrated that this methodology offers mtDNAreplacement with clinically applicable materials, and results in cellswith functional mitochondria that enable phenotypic recovery of themitochondria replaced cells (MirC).

Example VII: Inhibition of mTOR by Rapamycin Enhances Macropinocytosisof Exogenous Mitochondria in ρ(−) Cells

To determine a method for increasing the ability of a cell to undergoMirC, the mechanisms that regulate the macropinocytosis of exogenousmitochondria were investigated. Since ρ(−) cells are exhausted of ATP asa result of the reduced mitochondria, it was hypothesized that theintracellular energetic state of ρ(−) cells was similar to a starvedstate. To this effect, two molecular pathways were investigated:mammalian target of rapamycin complex 1 (mTORC1) and AMP-activatedprotein kinase (AMPK). mTORC1 is an essential sensor of amino acids,energy, oxygen, and growth factors, and a key regulator of protein,lipid, and nucleotide synthesis. AMPK is a sensor of AMP levels, and theactivation results in autophagy, mitochondrial biogenesis, glycolysis,and lipolysis. Both pathways are involved in uptake of extracellularnutrients.

As illustrated in FIG. 6D, to investigate the mechanism ofmacropinocytosis in ρ(−) cells, starvation was used to stimulateAMPK/mTORC1, while the “drugs” palmitic acid and rapamycin were used tospecifically stimulate mTORC1 activation, and suppress mTORC1,respectively. Rapamycin was added into the culture media at theconcentration of 50 ng/ml for 24 hours, and cells were exposed toglucose and essential amino acids free media without serum for 1 hour tosimulate starvation. Although palmitic acid (PA) was reported toactivate mTORC1 at a concentration of 200 μM in vivo, the titration ofPA for cultured fibroblasts showed the concentration of 50 μM and theduration of 24 hours was optimal based on the cellular viability. Theratio of phosphorylated AMPK to AMPK and phosphorylated p70 S6 kinase top70 S6 kinase, which is a downstream target of mTORC1, were examined byusing capillary electrophoresis, Wes™ (Protein Simple).

Treatment with PA or rapamycin demonstrated that although AMPK pathwaywas not significantly activated in ρ(−) cells (FIG. 6G and FIG. 6H), themTORC1 pathway was drastically suppressed in ρ (−) cells as measured bypS6/S6, at levels similar to starvation and rapamycin (FIG. 6E-FIG. 6F).These results demonstrated that mTORC1 represents an important targetfor macropinocytosis of mitochondria in ρ(−) cells.

Next, we examined the effects of rapamycin and palmitic acid uponmitochondria engulfment by treating the cells with rapamycin or palmiticacid simultaneously during the mitochondria co-cultivation. A scheme ofthe protocol is illustrated in FIG. 6I. Briefly, the NHDF recipientcells were transfected with the MTS-XbaI expression vector and culturedwith or without rapamycin, or with or without palmitic acid (PA).Puromycin selection for ρ(−) cells expressing the MTS-XbaI was performedafter 48 hours. On day 6, transfer of isolated mitochondria marked withDsRed from EPC100 cells was performed. On Day8, FACS analyses wereperformed to detect the donor mitochondria by measuring DsRed expressionin the NHDF recipient cells.

As shown in FIG. 6I-FIG. 6L, rapamycin treatment significantly enhancedthe engulfment of the DsRed-labeled isolated, exogenous mitochondria,whereas palmitic acid clearly suppressed it. These experiments wererepeated 4 times, and the positive fractions were summarized, whichindicated a statistically significant differences in rapamycin andpalmitic acid to ρ(−) cells (FIG. 6I and FIG. 6K). Notably, in both mocktransfection and add-on type mitochondrial transfer, there were nosignificant differences. In addition, the results showed that the effectof modulating mTORC1 activity only affected mitochondrial transfer ofρ(−) cells, and had no effect on “add on” or mock transfected cells,which indicated that this mechanism of transferring exogenousmitochondria was unique to the invention provided herein.

These results indicate that activation of mTORC1 by rapamycin duringmitochondrial transfer can enhance macropinocytosis of mitochondria.Further, these methods demonstrate that rapamycin, which is a clinicallyavailable drug, can be used to increase the efficiency ofmacropinocytosis for MirC generation.

Example VIII: mtDNA Replacement with Heteroplasmy Reversal inFibroblasts Derived from Patient with Leigh Syndrome

To investigate whether mitochondrial diseased cells could be correctedby using the in-vitro mtDNA replacement technique, primary fibroblasts(7SP) derived from a patient diagnosed with Leigh syndrome having amtDNA T10158C mutation were used as recipient cells (FIG. 7A). The sameprotocol described previously in NHDF cells was applied to 7SPfibroblasts. DNA sequencing of mtDNA in the EPC100 donor mitochondria atthe 10158th nucleotide was verified to be T (FIG. 7B, top), whereas the7SP fibroblasts has a mosaic of T in a major wave and C in a minor wave,indicating a heteroplasmy (FIG. 7B, bottom).

The kinetics of the content of mtDNA in 7S fibroblasts was almost thesame as in NHDF following the mitochondria replacement (FIG. 7C and FIG.7J). The time-lapse observation revealed that ρ(−) 7SP fibroblastsexhibited the same behavior with that of ρ(−) NHDF cells. In particular,accumulated aggregates of exogenous mitochondria upon the surface ofρ(−) cells became smaller and less over time and the intensity of DsRedin cytosol rapidly reduced suggesting an efficient engulfment into thecytosol and digestion in the cytosol, which was consistent with theresults generated using ρ(−) NHDF cells.

Importantly, the number of mtDNA copies following the mitochondriareplacement recovered to the same value with that of the original 7Sfibroblasts on day 12 (FIG. 7D). On the other hand, mock transfectant of7SP fibroblasts (add-on type mitochondria transfer) was unable to evenincrease the number of mtDNA copies in spite of the same cocultivationwith isolated mitochondria under the same conditions, which demonstratedpoor transfer of exogenous mitochondria (FIG. 7D, light gray bar).

Whether the mitochondria in 7S fibroblasts contained exogenous andhealthy mtDNA was examined by sequencing the mitochondrial genomefragment that included the 10158 nucleotide. As shown in FIG. 7E, themtDNA sequence of the 7SP cells changed from having a majority of mutantheteroplasmy at the 10158 nucleotide position (large wave of C and asmall wave of T) to a majority of wild-type mtDNA (large wave of T and asmall wave of C) in the recipient 7SP ρ(−) cells following mitochondriareplacement (FIG. 7E, bottom).

To generate quantitative information, a single nucleotide polymorphism(SNP) assay was performed to estimate the heteroplasmy generated usingthis technology. The ND3 region of mitochondrial DNA was amplified usinghmt10085-F primer (5′-CAACACCCTCCTAGCCTTACTACTAA-3′ (SEQ ID NO: 17)) andhmt10184-R primer (5′-GTCGAAGCCGCACTCGTA-3′ (SEQ ID NO: 20)), and theEPC100 specific probe (5′-ACATAGAAAAATCCACCC-3′ (SEQ ID NO: 18)) or the7SP specific probe (5′-CTACATAGAAAAATCCAC-3′ (SEQ ID NO: 19)) (FIG. 7F).The results indicated that the original hmt10158 heteroplasmy level in7SP fibroblasts was about 90% mutant mtDNA (FIG. 7G). The heteroplasmyin the 7SP cells that received the mitochondrial transfer (7SP ρ(−) Mt)exhibited as little as 10% heteroplasmy level on day 12 after thereplacement (FIG. 7G), while the mock transfectant (add-on typemitochondria transfer) did not significantly change the heteroplasmy andmaintained almost the same ratio or over 90% (FIG. 7H and FIG. 7I).These results indicated that the mitochondrial replacement technologyprovided herein is superior to the add-on mitochondrial transfer whichwas reported previously. The ρ(−) cells which went through theendonuclease treatment improved the heteroplasmy to about 75% with about80% reduction of the number of mtDNA copies.

Taken together these results demonstrate that the methods of generatinga mitochondrial replaced cell described herein that use MTS-XbaI topartially reduce endogenous mitochondria can be effectively used incells from a subject with a mitochondrial disease or disorder to improvethe heteroplasmy level and reduce the amount of mutant mtDNA.

Example IX: mtDNA Replacement in Fibroblasts Derived from Patient withLeigh Syndrome Yields Improved Cell Lifespan and Cell Metabolism

The functional activity of mitochondrial replaced 7SP fibroblasts wasevaluated. As shown in FIG. 8A and FIG. 8B, the proliferation ofmitochondrial replaced 7SP fibroblasts (ρ(−) Mt) cells was able torecover to levels equivalent to that of the original 7SP fibroblastsaround day 12.

In addition, the mitochondrial replaced 7SP fibroblasts (ρ(−) Mt) cellsdemonstrated a dramatic extension of lifespan, up to about the 63thpopulation doubling level (PDL) while the doubling time was over 120hours, which is the threshold of growth arrest (FIG. 8C). The cellsreceived the mtDNA replacement at about the 8th PDL and thereconstituted cells with the healthy mtDNA were able to continuedividing beyond the 55th PDL, which is thought to be the number of timesa normal human cell population will divide before cell division stops(i.e., the Hayflick limit). In contrast, the naïve 7S fibroblasts fellinto senescence at the 25th PDL (FIG. 8C). Thus, the experimentindicated the mtDNA replacement made a significant impact on theproliferation and lifespan of the mitochondrial diseased cells. Giventhat senescence increases with aged and cancer cells often involvemitochondrial dysfunction, this methodology might provide a crucial cluein rejuvenation and this might provide a basis for a novel strategy forcancer therapy as well as therapies for other age-related diseases.

The functional effect of mitochondrial transfer in 7S fibroblasts wasfurther evaluated by measuring the cell size (FIG. 8D). The mutation in7S fibroblasts in the coding sequence of the ND4 gene of Complex I inthe respiratory chain resulted in a disturbance of Complex I to transferelectrons coupled with its function to pump protons up from the matricesto intermembrane space. As a result, glycolysis was dominant to themitochondrial ATP production in 7S fibroblasts and resulted in thecompensatory adaptation to be bigger in cellular size to contain moremitochondria despite the damages and poor function (FIG. 8D). Relativeto PDL 15 (solid black line), by the PDL 25, the diameter of 7Sfibroblasts was about 1.5 times larger than that of NHDF, and theincrease in cell size doubled by PDL 35, and eventually the sizeincreased to about 3 to 8 times larger (FIG. 8D, left).

Consistent with the functional recovery of 7SP cells after the mtDNAreplacement, the notable increase in cell size observed in the early PDLin 7SP fibroblasts was inhibited following the mitochondria replacement(FIG. 8D, right). Moreover, the size of mitochondria replaced 7SP cells,which received the exogenous mitochondria at PDL 8, was maintainedthrough up to the 50th PDL (FIG. 8D, right). In addition, theconcentration of Citrate synthetase (CS) was two times more in 7SPfibroblasts by the 10th PDL than the CS concentration in NHDF cells,which is in agreement with the increase in size-up of 7SP fibroblasts(data not shown).

In order to confirm that the observed improvement in cell function aftermitochondria replacement was not due to contamination with other celltypes, a short tandem repeat (STR) assay was performed that candefinitely discriminate cells with different origins (FIG. 8E).Importantly, the patterns of STR in mitochondria replaced cells atdifferent time point were completely identical to that of the original7SP fibroblasts (FIG. 8E), which indicated no contamination.Furthermore, a RT-PCR revealed that the transfer of exogenousmitochondria derived from cells that express telomerase and E6, did nottransform the primary fibroblasts into cancer cells (FIG. 8F).

Taken together, these results demonstrated that mitochondrial transferof exogenous isolated mitochondria having wild-type mtDNA into 7SPcells, which are derived from a patient with Leigh Syndrome, increasedthe lifespan of 7SP cells, and improved the cellular function.Importantly, the transfer did not transform the mitochondrial replaced7SP cells into cancer cells.

Example X: Transfer of Exogenous Mitochondria to Fibroblasts Derivedfrom a Patient with Leigh Syndrome Yielded Functional Mitochondria

The functional effect of mitochondrial replacement in the 7SPfibroblasts was further evaluated by analyzing the cells' respiratoryfunction by using Oroboros 02k (FIG. 9A). Quantification of the resultsindicated that the basal respiration and ATP production (Free RoutineActivity) continued to decrease from the 10^(th) PDL to the 20th PDLafter the generation of a mitochondrial replaced cell and the maximumcapacity of electron transfer system kept the original levels of 7SPfibroblasts (FIG. 9B). By the 30th PDL after transfer of exogenousmitochondria, all three indices of the respiratory function (Routine,ETS, and Free routine activity) were elevated, and even surpassed thelevels of the original cells (FIG. 9B). These results indicated thatthere was a brief delay to reconstitute the electron transfer systemwith a healthy and non-mutated complex I following the mtDNAreplacement. Proton leakage showed the same kinetics with that of thenon-mitochondrial ATP production, which steadily improved from the earlyphase (FIG. 9B).

These results demonstrated that transfer of exogenous mitochondria intofibroblasts derived from a patient with a mitochondrial disease ordisorder can yield functional mitochondria.

Example XI: Transfer of Exogenous Mitochondria can Dissipate Chronic andSustained Reactive Oxygen Species (ROS) Generation

In order to characterize the properties of 7SP fibroblast-derived MirC,both a reperfusion and a starvation model under a culture condition wereused for 7SP fibroblast-derived MirC, the original 7SP fibroblast, andNHDF as a control. These stress conditions induce apoptosis in culturedcells, the extent of which can be quantified by AnnexinV as an earlymarker and propidium iodide (PI) as a late marker. Among environmentalinsults, the reperfusion injuries are mainly attributed to mitochondrialdysfunction. Cells predisposed to mitochondrial dysfunction due to mtDNAmutation are more fragile to the reperfusion injuries than healthycells.

Cells were seeded on 6 well plate at 1×10⁵ cells per well. The next day,600 μM H₂O₂ (FUJIFILM Wako Pure Chemical) was added to cells for thereperfusion model or essential amino-acid-free (“-EAA”) DMEM (FUJIFILMWako Pure Chemical) without serum was used as the culture media for thestarvation model. After 3 h H₂O₂ or 48 h starvation treatment, cellswere washed with PBS and collected to centrifugal tube. Annexin V-FITCand PI solution were added in cells and allowed to react for 30 minutesat room temperature protecting from light. Then cells were rapidlysubjected to FCM analysis using 488 and 561 nm laser lines. Fluorescencedata were collected using SH800 (Sony). The flow cytometry files wereanalyzed by using FlowJo software (TreeStar).

The results indicated that 7SP cells, which originate from a subjectwith Leigh syndrome, were highly susceptible to both forms of stress(i.e., H₂O₂ and starvation). As shown in FIG. 10A-10D, 7SP cells treatedwith H₂O₂ exhibited a significant increase in both early and lateapoptosis. In the reperfusion model (H₂O₂), NHDF did not exhibit anysignificant damages in the process of apoptosis based on AnnexinV and PIstaining (FIG. 10B-FIG. 10D). However, this mild reperfusion stressinduced apoptosis in 7SP fibroblasts. In contrast, the positivefractions of both AnnexinV and PI in 7SP fibroblast-derived MirC weresignificantly lower than the parental 7SP fibroblast, and near thelevels of NHDF cells (FIG. 10B-FIG. 10D). Importantly, there were nosignificant differences between 7SP fibroblast-derived MirC and NHDF,suggesting that the MirC regained the capability to tolerate this mildreperfusion damages.

The same trends as the reperfusion were recognized using the starvationmodel (FIG. 10E-FIG. 10H). Higher apoptosis in both an early and latephase was showed in 7SP fibroblasts, whereas 7SP fibroblast-derived MirCexhibited the almost same levels of apoptosis, basal values, as NHDF,which was significantly lower than those in the original 7SP fibroblasts(FIG. 10F-FIG. 10H). These results further confirm the mitochondrialreplacement method of the present invention improves the functionalrecovery of the recipient cell.

These results demonstrated that the transfer of exogenous mitochondriafrom a healthy cell into a cell with mutant mtDNA can improve thefunction of the recipient cell.

Example XII: Transfer of Exogenous Mitochondria into Recipient CellsReverted Early Stage Senescence-Associated Secretory Phenotype (SASP)

This example demonstrates that transfer of exogenous mitochondria intorecipient cells reverted early stage senescence-associated secretoryphenotype (SASP). A SASP consisting of inflammatory cytokines, growthfactors, and proteases is a characteristic feature of senescent cells.

To determine whether transfer of exogenous mitochondria into senescentcells could revert the SASP, the expression levels of the representativeSASP cytokines, IL-6 and IL-8, chemokine, CXCL-1, and growth factor,ICAM1 were quantitatively measured at the transcript levels for NHDF,7SP fibroblast, and 7SP fibroblast-derived MirC cells, whose PDLs werealmost the same, about 15 to 20 (FIG. 11). IL-6 was significantly higherin 7SP fibroblasts than those in NHDF and 7SP fibroblast-derived MirC,whereas the other three factors did not show any significant differenceamong these cells. At this PDL, 7SP fibroblasts did not exhibit atypical SASP, but only higher IL-6 expression, suggesting the earlyphase of senescence. Importantly, the process of the early-stagesenescence in this PDL of 7SP fibroblast-derived MirC could be reverted.

Taken together, these data demonstrate that mitochondria replacement isable to not only treat mitochondrial diseases with mutations of mtDNA,but also rejuvenate senescent cells, such as cells involved in an arrayof diseases, including neurodegenerative, cardiovascular, metabolic, andautoimmune diseases, and even cancers.

Example XIII: iPS Cells Generated from mtDNA Replaced Fibroblasts

To determine whether inducible pluripotent stem cells (iPSCs) could begenerated using cells derived from patients with a long deletion ofmtDNA, we attempted to build iPSC from 7SP fibroblasts using thestandard methods with Sendai virus carrying Oct3/4, Klf4, Sox2, andc-Myc (OKSM), which worked well for NHDF. A diagram of the protocoldesign is provided in FIG. 12A.

Alkaline phosphatase staining (AP staining), which detects iPSCscolonies at the early stage, demonstrated that the original 7SPfibroblasts-derived colonies seemed to be with crumbling appearances,whereas the mitochondria replaced 7SP fibroblasts-derived colonies weresolid on day 21 (FIG. 12B). In contrast, the ρ(−) 7SP fibroblasts thatdid not receive mtDNA replacement did not generate any colonies (FIG.12B). Several lines of iPS cells were able to be generated from themitochondria replaced 7S fibroblasts, as measured by AP staining (FIG.12C and FIG. 12D). The iPSC clones were stable in culture and exhibitedsimilar morphology between independent colonies (FIG. 12E).Immunohistochemistry staining confirmed the expression of the humanpluripotent stem cell markers SOX2, OCT3/4, NANOG, SSEA4, TRA1-81, andTRA1-60 on the mitochondria replaced 7SP fibroblasts-derived coloniesoverexpressing OKSM (FIG. 12F).

The iPSC generated by the methods described herein were further comparedto the commercially available KYOU-DXR0109B Human Induced PluripotentStem (IPS) Cells [201B7]. Importantly, the mitochondria replaced 7SPfibroblasts showed the same level of efficiency with the iPS generationwith that of healthy fibroblasts. Additionally, in agreement withprevious studies, qPCR of 12S-rRNA, normalized to nuclear (3-actin,demonstrated that the iPS cells generated by mitochondrial replaced 7SPfibroblasts exhibited half of the mtDNA contents relative to control,and the mtDNA levels were similar to that of the 201B7 iPSC standard(FIG. 12G).

Moreover, the hmt10158 heteroplasmy level was less than 10% in thegenerated iPSCs (FIG. 12H). Quantification of the absolute mtDNA copynumber confirmed the reduced level of mtDNA and reduction in mutantmtDNA (FIG. 12I).

These results demonstrate that iPSCs can be generated using themitochondrial replacement technology provided herein, and could beapplicable in the clinical field because this whole procedure used onlymaterials adaptable to clinics.

Example XIV: Mitochondria Replacement of Mitochondria from Donor CellAlters Recipient Cell's Lifespan Cell

This example demonstrates that mtDNA replacement can alter the lifespanof the recipient cell. In order to validate the hypothesis that themitochondria replacement can rejuvenate senescent cells, two models wereestimated in respect of cell cycle capabilities, such as doubling timeand PDL at the growth arrest.

NHDFs and TIG1 embryonic lung cells with early PDL (around 5 to 10,called “young”) and late PDL (around 40 to 45, called “old”) wereutilized to design the models. One model involved young cells replacedwith mitochondria derived from old cells, designated as “02Y,” andanother model involved old cells replaced with mitochondria derived fromyoung cells, designated as “Y20” (FIG. 13A).

The extent of the exchange of mtDNA was evaluated by TaqMan SNPgenotyping assay, based on the difference of the single nucleotide ofmtDNA at the 16145 position between NHDF and TIG1, which are A and G,respectively (FIG. 13B). NHDF-derived MirC clearly showed that more than90% of endogenous mtDNA (hmt16145-A) was replaced with TIG1-derivedmtDNA (hmt16145-G) (FIG. 13C). The small percentage of hmt16145-Adetected in the parental TIG1 cells was considered to be the backgrounderror (FIG. 13C).

In addition, the Y20 model clearly demonstrated a regain of the lifespanin old cells to around 65 PDLs (FIG. 13D). Control old cells and mocktransfectant showed the growth arrest at 55 PDLs, which is consistentwith the Hayflick limit. On the other hand, 02Y demonstrated the reducedlifespan of young cells at about 45 PDLs (FIG. 13E). The difference,around 10 PDLs, in both models could be attributed to exogenous mtDNA.These results demonstrate that transfer of exogenous mitochondria from ayoung cell to an old cell can rejuvenate cells.

Example XV: Optimization of Mitochondria Replaced Cell (MirC) from HumanPrimary T Cells Using mRNA Transfection

This example describes the generation of mitochondria replaced Cell(MirC) from human primary T cells by using mRNA transfection.

Prior to the experiments, use of human primary T cells were approved byour institutional ethical committee. Peripheral blood was drawn from ahealthy volunteer and centrifuged using percoll with a specific gravityof 1.077 at 400 g for 35 minutes at 20 degree to separate lymphocytes.Isolated lymphocytes of 1×10⁶ cells per ml were seeded onto a 96-wellflat plate coated with anti-CD3 and anti-CD28 antibodies. The plate wasprepared by incubating with 5 μg/ml of anti-CD3 and 1 μg/ml of anti-CD28of overnight and pre-warmed at 37° C. 2 hours prior to the seeding. Onthe next day of the seeding, IL-7 and IL-15 were added to the medium ata concentration of 20 μg/ml and 10 μg/ml, respectively. Medium waschanged every third day with IL-7 and IL-15 at the same concentration asthe initial addition.

Transfection was performed using the MaxCyte electroporator, which meetsthe standard of GMP/GCP, according to the manufacturer's protocol. mRNAwas created according to the manufacturer's protocol in mMESSAGEmMACHINE T7 Ultra Kit (Thermo Fisher), with slight modifications.Briefly, a DNA template for mRNA was prepared from the plasmid carryingthe DNA sequence without refining the fragment following endonucleasedigestion, in order to reduce the possibility of mixing RNase up aslower as possible (FIG. 14A).

The results indicated that the non-refining DNA template for mRNAcreation of EGFP led to nearly a 100% gene transfer efficiency with highexpression and high viability at 24 hours after the gene transfection(FIG. 14B and FIG. 14C). No antibiotics selection was required usingthis method due to the high transfection efficiency. In addition,transfection of MTS-XbaIR resulted in a reduction in mitochondrialmembrane potentials, which could be attributed to the reduction of inendogenous mtDNA (FIG. 14D).

In order to determine the optimal protocol with respect to the timing ofisolated mitochondria co-incubation, fluorescent images of human primaryT cells that received mRNA of GFP by electroporation using MaxCyte ATXwere taken over an 8-day period, as indicated (FIG. 14E). Thefluorescent images of control electroporated cells (FIG. 14F, upperpanels) where cells were transfected with a GFP plasmid showed similarkinetics as that in fibroblasts. Expression peaked at day 2 anddisappeared by day 8. In contrast, cells that received MTS-GFP mRNA(FIG. 14F, lower panel) showed higher expression within 4 hourspost-electroporation and an earlier disappearance on day 6 than those incells transferred with the plasmid.

The protein expression of GFP in cells receiving the MTS-GFP mRNA wasevaluated by western blotting analysis using a capillaryelectrophoresis. The peak expression occurred at day 4, and expressionwas lost by day 6, as illustrated in the western blot (FIG. 14H) andquantified in FIG. 14G. Quantification of kinetics of XbaIR transcriptlevels were performed by qPCR and revealed that the transcriptexpressions of the endonuclease were quite highest at 4 hours post-genetransfer (FIG. 14I). The XbaIR transcript levels rapidly decreased byday 2, and were negligible by day 6 (FIG. 14I). The mitochondrialcontents were estimated by quantifying 12S rRNA (FIG. 14J), anddemonstrated that mitochondria decreased to about 30% by day 2, and wasmaintained at less than 20% throughout the length of the experiment.

Taken together, these results demonstrate that mRNA transfection of anendonuclease, such as XbaI, fused to an MTS can efficiently degrade thehost mtDNA, and can be used to generate a mitochondria replaced Cell(MirC) from human primary T cells.

Example XVI: Generation of Mitochondria Replaced Cell (MirC) from HumanPrimary T Cells Using mRNA Transfection

After determining the optimal time point for performing mitochondrialtransfer in human primary T cells from Example XV, mitochondriaco-incubation was performed on day 7 to prevent digestion of exogenousmtDNA by any remaining endonuclease. A scheme of the MirC protocol forhuman primary T cells is shown in FIG. 15A.

In order to determine the heteroplasmy of mtDNA in the recipient humanprimary T cells following mitochondria replacement, the difference ofmtDNAs between the donor mitochondria and the recipient cells wasdetermined by TaqMan SNP genotyping assay. Sequencing of the D-loop ofmtDNA in normal human primary T cells and EPC100 (mitochondria donorcells) showed a difference in 2 nucleotide positions (nucleotides 218and 224 mtDNA), which were C/C and T/T for T cells and EPC100 cells,respectively (FIG. 15B). To delineate the standard curve for TaqMan SNPgenotyping assay, the fragment of variable region encompassing the 218and 224 nucleotides of mtDNA was subcloned into pBluescript SK(−). Thepolymorphic nucleotides were mapped on the human Cambridge ReferenceSequence, and primers and probes were designed to amplify and target thedesired region of the D-loop, where the probes had FAM and VICfluorophore (FIG. 15C). Using TaqMan polymerase with 5′ exonucleaseactivity, qPCR was carried out and threshold cycle (Ct value) wasdetermined, which was fit to a standard curve created using severaldifferent copy numbers of the above mentioned plasmids for eachsequence. Following the somatic mitochondria replacement, the origin ofEPC100 mtDNA dominated in human T cells on both day 7 and day 12,whereas mock transfectants that received electroporation without geneticmaterial and were coincubated with isolated mitochondria at the sameprotocol as MirC exhibited the exogenous origin of mtDNA less than 10%on day 7, and the background levels on day 12 (FIG. 15D). Thisdemonstrated that the MTS-XbaIR mRNA facilitated efficient mitochondrialtransfer in human primary T cells.

Next, to evaluate the effect that the mitochondrial transfer had on thefunction of the MirC human T cells, respirometry experiments wereperformed using Oroboros 02k. The results demonstrated a recovery of ATPproduction and coupling efficiency in human T cell-derived MirC on day7, whereas ρ(−) human T cells that were generated by XbaIR mRNA transferwith electroporation maintained the loss of ATP production throughoutthe experiment (FIG. 15E). Representative raw data usingcoupling-control protocol (CCP) are depicted in FIG. 15F and FIG. 15G,and show that MirC T cells are able to restore mitochondrialrespiration.

These results demonstrated that human primary T cells are capable ofmitochondria replacement to generate MirC using GMP gradedelectroporator, such as the electroporator produced by MaxCyte Inc.

Example XVII: Generation of Mitochondria Replaced Cell (MirC) from MousePrimary T Cells Using mRNA Transfection

Further characterizations of T cell-derived MirC were executed formurine T cells. The isolation of murine T cells from suspensionsolutions obtained from the spleen was performed using the EasySep MouseIsolation Kit (STEM CELL Technologies, Inc.), which provides highlypurified T cell population by negative selection using magnets. Isolatedmurine T cells (1×10⁶ cells per ml) were seeded onto 96-wells plate withDynabeads mouse T-Activator CD3/CD28 (Invitrogen, Inc.) at abead-to-cell ratio of 1:1 and recombinant IL-2 at 30 U/ml. The medium tocultivate murine T cells was determined with respect to cell growth andCD3 expression, and RPMI1640 was found to be superior to TexMACS (FIG.16A). For example, viability and total cell number were higher in cellscultured in RPMI1640, as compared to TexMACS. The medium was changedevery third or fourth day.

Next, electroporation of murine T cells was performed using theNucleofector machine and mRNA. The kinetics of GFP expression followingmRNA transfer were similar to human T cells (FIG. 16B). For example, 6hours after electroporation of MTS-GFP mRNA, almost all of the cellswere found to strongly express GFP (FIG. 16B). This demonstrated thatthe MTS-GFP was transfected with high efficiency. The intensity of GFPrapidly declined with time, and eventually disappeared on day 6following the electroporation (FIG. 16B).

Transfection of the MTS-XbaI mRNA indicated that murine T cellsexhibited a milder decline of XbaIR transcript expression relative tohuman T cells, and persisted even at a low level on day 6 (FIG. 16C).Quantification of 12S rRNA levels as a surrogate marker for mtDNA,indicated that the murine mtDNA persisted even on day 6 at about 40% ofthe control (FIG. 16D). Next, coincubation of Ds-Red labeled exogenousmitochondria and ρ(−) murine T cells on day 5 was performed (FIG. 16E).Despite the longer persistence of XbaIR, and lower levels of endogenousmtDNA reduction, relative to human T cells, FACS analysis of engulfedfluorescence-labeled mitochondria 48 hours following the co-incubationwith isolated mitochondria revealed a significant positive fraction(9.73%) of T cells expressing exogenous mitochondria (FIG. 16F). Thepercentage of positive cells expressing exogenous mitochondria was evenhigher than that in fibroblast experiments, demonstrating that thisprotocol for murine T cells could be optimal to generate T cell-derivedMirC.

Example XVIII: Transfer of Exogenous Mitochondria to T Cells RevertedSenescence

This example demonstrates that mitochondria replacement was successfulin murine T cells, and rejuvenated senescent T cells.

To evaluate whether exogenous mitochondria could be successfullytransferred to generate murine T cell-derived MirC, mtDNA heteroplasmylevels were of BL6 (recipient) cells and NZB (donor) cells.Two-consecutive polymorphisms at 2766 and 2767 mtDNA for ND1 wasverified (FIG. 17A). Specifically, the BL6 mitochondria contained AT atpositions 2766 and 2767 of mtDNA, whereas the NZB mitochondria containedGC at the same positions. A primer set and two probes were designed todiscriminate the polymorphism using a different fluorophore for each ofthe GC and AT polymorphisms (FIG. 17B). In addition, two separateplasmids were generated to express the GC and AT polymorphisms,respectively, and a standard curve was generated to facilitate thequantitative estimation of heteroplasmy in MirC.

Quantification of the mitochondria replacement (XbaIR Mt) in BL6 cellsthat were transfected with MTS-XbaI and co-incubated with mitochondriaisolated from NZB mice, demonstrated an overwhelming domination ofexogenous mtDNA, whereas mock transfectants that were coincubated withisolated mitochondria following electroporation under the absence ofmRNA of the endonuclease did not engulf any exogenous mtDNA (FIG. 17C).This result demonstrated that T cells are permissive to mitochondriareplacement.

Because the results described herein demonstrated fibroblast-derivedMirC could undergo rejuvenation in vitro (FIG. 13), T cell-derived MirCwas also examined for rejuvenation potential. Recipient cells from oldmurine T cells were prepared from the spleen of mice (C57BL/6) that weremore than 80 weeks old, and donor murine mitochondria were isolated fromthe liver of mice (C57BL/6) around 10 weeks old. Telomere length hasbeen reported to shorten with age. Therefore, telomere length wasmeasured by using Absolute mouse Telomere Length Quantification qPCRAssay Kit (ScienCell, Inc.). Following the treatment of old murine cellswith the MTS-XbaIR mRNA and co-incubation with exogenous mitochondriafrom the young donor cells to generate the MirC (Young to Old: YtoO),telomere length was observed to have a 1.7-fold increase in length,relative to the original old T cells (FIG. 17D). This demonstrated thatthe mitochondrial replaced cells exhibit characteristics ofrejuvenation.

In addition, SASP was evaluated using the same representative set ofmakers described previously (FIG. 11). The measurement of CXCL1, ICAM1,IL-6, and IL-8 revealed that Murine T cell-derived MirC decreased IL-6and CXCL-1, and showed no change in ICAM-1 and IL-8 (FIG. 17E). Theseresults indicated a decrease in SASP for the MirC T cells.

Moreover, senescent T cells have been found to exhibit higher DNA damageresponse (DDR), compared with young T cells. Therefore, DDR was measuredusing the histone 2 A (H2A) phosphorylation antibody, for the MirC andthe original T cells. The results indicated that the positive fractionfor DDR was lower in the MirC (1.53%), compared with the original Tcells (4.75%) (FIG. 17F). Thus, the MirC T cells had lower levels ofDDR, indicating a reversal of senescent behavior.

These in vitro results support that the somatic mitochondria replacementwas verified in the MirC mtDNA, and the replacement resulted in numerouschanges that were indicative of a reversal of senescence in the MirC Tcells.

Example XIX: Tumor Growth is Mitigated by Adoptive Cell Transplantation(ACT) Using MirC Derived from Old T Cells Containing ExogenousMitochondria from Young Mice

To examine the functional potential of the mitochondria replacement thatrejuvenated senescent cells, an adoptive cell transplantation (ACT)experiment was performed. The AE17 mesothelioma cell line derived fromthe peritoneal cavity of C57BL/6J mice injected with asbestos fibers wasused to develop tumor formation in mice. Previous experiments using thismodel have shown that tumor growth is mitigated by ACT of youngsyngeneic T cells, but not by ACT of old syngeneic T cells (Jackaman etal. Oncolmmunology 2019; 8(4): 1-16).

To determine whether the rejuvenated old T cells, generated bytransferring isolated exogenous mitochondria from a young mouse to a Tcell from an old mouse, exhibited functional activity, AE17 cells weresubcutaneous injected into three groups of old mice (Group 1: old micewith ACT of T cells from young mouse; Group 2: old mice; or Group 3: oldmice with ACT of MirC derived from a T cell of an old mouse transferredwith exogenous mitochondria from a young mouse (FIG. 18A). The old Tcell-derived MirC were evaluated for their capability to suppress thetumor growth. C57BL/6 mice aged 22 to 24 months were utilized in the ACTexperiment. The young mice used in the experiment were 2 to 3 months oldmice. In addition of body weight measurements, tumor growth was measuredusing NIH image of photographs taken every 3 days (FIG. 18B). AE17inoculation was executed on day −14 with 2×10⁶ cells suspended in 100 μLMatrigel, and the day of T cell transfer was considered to be day 0. Onday 0, 2×10⁶ cells of either young T cells or old T cell-derived MirCwere intravenously injected into tumor-bearing mice. On the same day,recombinant IL-2 (2 μg) was intraperitoneally injected once, followed bytwo more injections on day 2 and day 3.

The body weight in each group did not show significant differences (FIG.18C). However, the tumors were attenuated in both the Group 1 mice (oldmice with young T cells) the Group 3 mice (old mice with old Tcell-derived MirC), whereas the tumors steady grew in the Group 2 (mock)mice (FIG. 18D). The relative mean masses showed similar trends as theindividual mice, demonstrating that the MirC behaved like the young Tcells (FIG. 18E).

To verify the presence of infused T cells in the animals, T cellsderived from GFP transgenic mice were transplanted into the syngeneicC57BL/6 mice, the peripheral blood and the spleen were examined to trackthe donor cells (FIG. 18F). A two-dimensional plot with FSC versus FL-1to detect GFP fluorescence was generated to clarify the rare population.Negative controls using C57BL/6 mice (left upper panel), and positivecontrols using GFP transgenic mice (left lower panel) were generated forboth the peripheral blood and the spleen (FIG. 18G). The definitivepopulation of T cells expressing GFP fluorescence were recognized inboth samples, although the fractions were 0.057% and 0.9% in theperipheral blood and the spleen, respectively (FIG. 18G). Thetransferred T cells in this protocol were detected on day 6 followingthe transplantation (FIG. 18H), which validated that this protocol couldbe used to evaluate the capability of the transferred cell. In addition,the percentage of chimerism following infusion of the exogenous T cellswas found to increase when a greater amount of cells were infused (FIG.18I).

These results clearly demonstrate that ex vivo MirC generation usingmitochondria from young mice into T cells from an old mouse caneffectively function in vivo, and reduce tumor burden at similar levelsas T cells from young mice.

Example XX: Hematopoietic Stem Cells are Capable of MirC Generation

To date, gene transfer methods for hematopoietic stem cells have mainlyinvolved the use of viral vectors, because the targets were mainlygenetic disorders that require a sustainable gene expression of thedeficient gene. Consequently, electroporation is not used in currentprotocols of gene transfer of hematopoietic stem cells because of theneed to generate a permanent gene expression. In contrast, the objectiveof the mitochondrial replacement technology provided herein is toachieve temporal high expression of the exonuclease.

Based on the experiments for fibroblasts and T cells, the condition ofNucleofector/electroporation with mRNA was adjusted, and severalconditions were examined for murine fetal liver-derived Sca-1 positivecells (FIG. 19A), which are considered to be an enriched population forhematopoietic stem cells (HSCs). Among several conditions, threeconditions (program X-001, Y-001, and T-030 that are code number in themachine provider) were evaluated by immunofluorescence and cellviability (FIG. 19A). The experimental conditions were termed MTS-GFP1,2, and 3 according to the program that was used (program X-001, Y-001,and T-030, respectively).

Further examinations were performed by FACS analysis for the meanfluorescent intensities (MFI) on dayl following the electroporation withmRNA of GFP (FIG. 19B). The results indicated that the optimal conditionwas the X-001 program (MTS-GFP1) because although the right shift of MFIin the condition was little, it was significant compared with the others(FIG. 19B). Murine bone marrow-derived Sca-1 cells were coincubated withmitochondria isolated from the syngeneic murine cells that are a stablegene-modified cell line expressing DsRed fluorescence. 3-D fluorescentimaging of the bone marrow-derived Sca-1 cells 48 hours after theco-incubation showed that the exogenous mitochondria were engulfed (FIG.19C). The mitochondrial transfer efficiency was estimated by FACSanalysis for DsRed fluorescent axis, and revealed that a subpopulationof about 10% of the Sca-1 exhibited a right ward shift of thefluorescent, suggesting that BM-derived Sca-1 positive cells couldundergo somatic mitochondria replacement (FIG. 19D). However, thetransfer of exogenous mitochondria in the MTS-GFP expression cellswithout depletion of endogenous mitochondria was too low for clinicalapplication.

Next, we examined whether this mitochondria replacement procedure viageneration of ρ(−) cells using MTS-XbaIR mRNA transfer could beapplicable to hematopoietic stem cells (FIG. 19E). The Realhematopoietic stem cell population is considered as c-kit⁺, Sca-1⁺,Lineage⁻, CD34⁻ (called as KSLC) that is around 0.005% in the whole bonemarrow cells (Wilkinson, A. C. et al. Nature, 571(7763):117-121 (2019)).Following FACS sorting for KSL cells from murine bone marrow-derivedcells (FIG. 19F), the KSL cells were cultivated for 5 days in thepresence of stem cell factors and TPO with polyvinyl alcohol (PVA).Macroscopically, the KSL cells maintained the morphology and exhibited ashort doubling time of 19 hours (FIG. 19G).

The heteroplasmy changes were evaluated using the TaqMan SNP genotypingassay, as described above. A scheme of the assay is shown in FIG. 19H.Murine KSLC-derived MirC demonstrated that the exogenous mtDNA withpolymorphism in NZB was 99.9% on day 6 following the endonuclease mRNAtransfer with electroporation (FIG. 19I), which indicated that theexogenous mtDNA almost completely replaced the endogenous mtDNA ofCL57BL/6. These results demonstrated that hematopoietic stem cells arepermissive to this technology to generate MirC.

Example XXI: Droplet Digital PCR (ddPCR) for Measurement of mtDNA andHeteroplasmy

This example demonstrates that mitochondrial DNA (mtDNA) can be assayedfor the presence of a specific mtDNA sequence, such as a mutation inmtDNA, using digital PCR (dPCR). Droplet Digital PCR (ddPCR) is a methodfor performing digital PCR that is based on water-oil emulsion droplettechnology.

Primary skin fibroblasts derived from patients with mitochondrialdisease were analyzed. The patient information is provided below inTable 1.

TABLE 1 Patient information Sample Disease Mutation Age Sex InheritanceBK01 MELAS mtDNA A3243G 30 Male Mother (mt-tRNA) BK02 Leigh mtDNAT10158C 6 Female De Novo Syndrome (Complex I • MT-ND3) BK04 Leigh mtDNAT9185C 1 Female N.D. Syndrome (Complex V • MT-ATP6)

Cells from the target population were encapsulated into droplets at aconcentration of one cell per droplet with the PCR mixture includingprimers and probes. Cell density was optimized to generate a single cellin a single droplet, and the fibroblasts were finally diluted in 1×10⁶cell/mL for ddPCR. After single-cell encapsulation, cell lysis andamplification of the target sequence were performed within the droplets.The number of droplets with a fluorescent signal indicated the number ofcells carrying the target or reference gene.

Briefly, a 20× primer/probe mix was prepared as described below in Table2. The standard ddPCR master mix was a 25 μL mix that includes theaforementioned primer/probe mix, template DNA and 2×ddPCR super mix.

TABLE 2 dPCR Primer and Probe Mix 20× Primer/Probe Mix Volume (μL) per100 μL 100 μM F1 primer 10 100 μM R1 primer 10 100 μM labeled probe 5PCR grade water 75

TABLE 3 dPCR Reaction Master Mix Reagent Volume (μL) per 25 μL Reaction2× ddPCR super mix 12.5 20× Primer/Probe Mix 1.25 Template (100 ng/μL) 1PCR grade water 10.25

Samples were loaded into an 8-chamber cartridge using 20 μL of theprepared qPCR sample followed by 70 μL of droplet generation oil in theadjacent wells. A rubber gasket was stretched across the top of thechambers to ensure a vacuum seal. Each 8-chamber cartridge was loadedonto the QX100 droplet generator producing 20,000 droplets per sample.Using a 50 μL multichannel pipette, 40 μL of the generated droplets weretransferred to a 96-well plate and heat sealed with pierceable foil. Theplate was placed in a thermal cycler using standard 2-step qPCR thermalcycling conditions with a 50% (3° C./sec) ramp rate. Prior to runningthermal cycling conditions, primer/probe sets were optimized using atemperature gradient to optimize the anneal/extend temperature.

TABLE 4 dPCR Cycling Conditions dPCR Cycling Conditions Temp (° C.) Time(sec) Initial Hot Start/denaturation 95 600 Steps 1-2 are repeatedthrough 40 cycles Step 1 94 30 Step 2 60 60 Step 3 98 600 Step 4 12infinity

Following thermal cycling, the plate was loaded onto the QX100 dropletreader and end-point reactions were analyzed. Poisson statisticalanalysis of the numbers of positive and negative droplets yieldsabsolute quantitation of the target sequence.

Before examining diseased cells, the specificity for the probes to bedesigned for a mutated sequence and the sensitivity for the probes to bedesigned for a non-mutated sequence were evaluated by using normal humandermal fibroblasts (NHDF cells) that have a non-mutated sequence (thesame as Cambridge Reference Sequence) (FIG. 20A-FIG. 20C). The dots inleft lower area indicated no cells in the droplet. Evaluation of thethree different probe sets clearly detected the non-mutated sequence(lower right in BK01 (FIG. 20A), upper left in BK02 (FIG. 20B), andupper left in BK04 (FIG. 20C)), and did not detect the mutant sequence(upper left in BK01 (FIG. 20A), lower right in BK02 (FIG. 20B) and BK04(FIG. 20C)).

ddPCR of fibroblasts obtained from BK01 indicated a few percentage ofdouble positive population, and the majority was cells with homoplasmyof mutated mtDNA (FIG. 20D). There was no significant population withhomoplasmy of non-mutated mtDNA in a single cell. In addition, BK02showed a minor portion of double positive cells, which indicated aheteroplasmy in a single cell level, defined as microheteroplasmy (FIG.20E). The results from BK02 revealed a major population of homoplasmy ofmutant mtDNA, and no population with homoplasmy of non-mutated mtDNA wasnot recognized.

Taken together, these results demonstrated that homoplasmy andheteroplasmy can be accurately, and quantitatively evaluated at a singlecell level. In addition, the results demonstrate that the mtDNA of asubject with mitochondrial disease can be accurately measured, whichcould be useful for evaluating therapeutic compositions prior totransplantation in a subject or monitoring the mtDNA content prior toand/or after therapy.

Example XXII: MtDNA Replacement in Recipient Hematopoietic Stem orProgenitor Cells (HSPCs) from Donor cGMP Manufactured Bone-MarrowDerived Mesenchymal Stromal Cells (BM-MSCs)

This example demonstrates that hematopoietic stem or progenitor cells(HSPCs) can be ex vivo modulated using the mtDNA replacement methodsprovided herein for therapy.

Modulation of HSPCs can be performed ex vivo in connection with a stemcell transplant. Briefly, peripheral blood stem cells are mobilized anda blood sample is obtained from the patient. Peripheral hematopoieticstem or progenitor cells (HSPCs), e.g., CD34⁺ cells are isolated andsent to a manufacturing facility. At the manufacturing facility, themitochondria is partially depleted according to the methods providedherein.

Donor mitochondria are isolated using current Good ManufacturingPractice (cGMP) manufactured bone-marrow derived Mesenchymal StromalCells (BM-MSCs) obtained from a cell repository (e.g., WaismanBiomanufacturing). The initial bone marrow aspirates are collected withfull informed consent and in compliance with federal regulations (e.g.,21 CFR 1271). The aspirates are processed under cGMPs and banked at anearly passage for subsequent expansion.

The donor mitochondria from the BM-MSCs are transferred to culturedHSPCs, changing the heteroplasmy. The modified HSPCs are sent back tothe medical center for autologous transplantation (i.e., into the samesubject that the HSPCs were isolated). Prior to transplantation thepatient receives minimal treatment that can include a non-myeloablativeregimen, such as partial irradiation or sublethal dose of anti-cancerdrugs, such as busulfan. The modified HSPCs, only containing theallogenic donor mitochondria, are transfused back into the patient.

This example demonstrates that HSPCs can be ex vivo modulated using themtDNA replacement methods provided herein for therapy that does notinvolve transplantation of allogenic HSPCs.

The embodiments described above are intended to be merely exemplary, andthose skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, numerous equivalents ofspecific compounds, materials, and procedures. All such equivalents areconsidered to be within the scope of the invention and are encompassedby the appended claims.

1. A method of generating a mitochondria replaced cell, comprising: (a)contacting a recipient cell with an agent that reduces endogenous mtDNAcopy number (b) incubating the recipient cell for a sufficient period oftime for the agent to partially reduce the endogenous mtDNA copy numberin the recipient cell; and (c) co-incubating (1) the recipient cell fromstep (b) in which the endogenous mtDNA has been partially reduced, and(2) exogenous mitochondria or exogenous mtDNA from a healthy donor, fora sufficient period of time to non-invasively transfer the exogenousmitochondria or the exogenous mtDNA into the recipient cell, therebygenerating a mitochondria replaced cell.
 2. A method of treating asubject in need of mitochondrial replacement, a subject having orsuspected of having an age-related disease, or a subject having amitochondrial disease or disorder, comprising: (a) generating amitochondria replaced cell ex vivo or in vitro, comprising the steps of:(i) contacting a recipient cell with an agent that reduces mtDNA copynumber; (ii) incubating the recipient cell for a sufficient period oftime for the agent to partially reduce the mtDNA copy number in therecipient cell; and (iii) co-incubating (1) the recipient cell from step(ii) in which the endogenous mtDNA has been partially reduced, and (2)exogenous mitochondria from a healthy donor, for a sufficient period oftime to non-invasively transfer exogenous mitochondria into therecipient cell or exogenous mtDNA from a healthy donor, for a sufficientperiod of time to non-invasively transfer exogenous mtDNA into therecipient cell, thereby generating a mitochondria replaced cell, therebygenerating a mitochondria replaced cell; (b) administering atherapeutically effective amount of the mitochondria replaced recipientcell from step (a) to the subject in need of mitochondrial replacement.3-4. (canceled)
 5. The method of claim 1 or 2, wherein (a) the exogenousmitochondria comprises: (i) a functional mitochondria; (ii) wild-typemtDNA; (iii) isolated mitochondria, wherein the isolated mitochondria isoptionally an intact mitochondria; and/or (iv) allogeneic mitochondria;and (b) the endogenous mtDNA: (i) encodes for a dysfunctionalmitochondria; (ii) comprises mutant mtDNA; (iii) comprises mtDNAassociated with a mitochondrial disease or disorder; (iv) isheteroplasmic; or (v) comprises wild-type mtDNA; and/or (c) theendogenous mitochondria is dysfunctional. 6-13. (canceled)
 14. Themethod of claim 1 or 2, wherein the agent that reduces endogenous mtDNAcopy number is selected from the group consisting of a polynucleotideencoding a fusion protein comprising a mitochondrial-targeted sequence(MTS) and an endonuclease, a polynucleotide encoding an endonuclease,and a small molecule, wherein the small molecule is optionally anucleoside reverse transcriptase inhibitor (NRTI); wherein thepolynucleotide is optionally comprised of messenger ribonucleic acid(mRNA) or deoxyribonucleic acid (DNA); wherein the recipient celloptionally transiently expresses the fusion protein; wherein theendonuclease is optionally selected from the group consisting of XbaI,EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), andtranscription activator-like effector nuclease (TALEN); wherein the MTSoptionally targets a mitochondrial matrix protein; and wherein themitochondrial matrix protein is optionally selected from the groupconsisting of cytochrome c oxidase subunit IV, cytochrome c oxidasesubunit VIII, and cytochrome c oxidase subunit X. 15-20. (canceled) 21.The method of claim 1 or 2, wherein the agent that reduces endogenousmtDNA copy number reduces (a) about 5% to about 99% of the endogenousmtDNA copy number; (b) about 30% to about 70% of the endogenous mtDNAcopy number; (c) about 50% to about 95% of the endogenous mtDNA copynumber; (d) about 60% to about 90% of the endogenous mtDNA copy number;or (e) mitochondrial mass. 22-29. (canceled)
 30. The method of claim 2,wherein (a) the subject in need of mitochondrial replacement has adysfunctional mitochondria; a disease selected from the group consistingof an age-related disease, a mitochondrial disease or disorder, aneurodegenerative disease, a retinal disease, diabetes, a hearingdisorder, a genetic disease; or a combination thereof, wherein theneurodegenerative disease is optionally selected from the groupconsisting of amyotrophic lateral sclerosis (ALS), Huntington's disease,Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, CharcotMarie Tooth disease and leukodystrophy, and wherein the retinal diseaseis optionally selected from the group consisting of age-related maculardegeneration, macular edema and glaucoma; (b) the age-related disease isselected from the group consisting of an autoimmune disease, a metabolicdisease, a genetic disease, cancer, a neurodegenerative disease, andimmunosenescence, wherein the metabolic disease is optionally diabetes,wherein the neurodegenerative disease is Alzheimer's disease, orParkinson's disease, and wherein the genetic disease is optionallyselected from the group consisting of Hutchinson-Gilford ProgeriaSyndrome, Werner Syndrome, and Huntington's disease; (c) themitochondrial disease or disorder is caused by mitochondrial DNAabnormalities, nuclear DNA abnormalities, or both, wherein themitochondrial disease or disorder caused by mitochondrial DNAabnormalities is optionally selected from the group consisting ofchronic progressive external ophthalmoplegia (CPEO), Pearson syndrome,Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrialdiabetes, Leber hereditary optic neuropathy (LHON), LHON-plus,neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP),maternally-inherited Leigh syndrome (MILS), mitochondrialencephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS),myoclonic epilepsy and ragged-red fiber disease (MERRF), familialbilateral striatal necrosis/striatonigral degeneration (FBSN), Luftdisease, aminoglycoside-induced Deafness (AID), and multiple deletionsof mitochondrial DNA syndrome, and wherein the mitochondrial disease ordisorder caused by nuclear DNA abnormalities is selected from the groupconsisting of Mitochondrial DNA depletion syndrome-4A, mitochondrialrecessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinalencephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS),DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathydysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstemand spinal cord involvement and lactate elevation (LBSL), co-enzyme Q10deficiency, Leigh syndrome, mitochondrial complex abnormalities,fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC)deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenasecomplex deficiency (PDHC), pyruvate carboxylase deficiency (PCD),carnitine palmitoyltransferase I (CPT I) deficiency, carnitinepalmitoyltransferase II (CPT II) deficiency, carnitine-acyl-carnitine(CACT) deficiency, autosomal dominant-/autosomal recessive-progressiveexternal ophthalmoplegia (ad-/ar-PEO), infantile onset spinal cerebellaratrophy (IOSCA), mitochondrial myopathy (MM) spinal muscular atrophy(SMA), growth retardation, aminoaciduria, cholestasis, iron overload,early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).31-45. (canceled)
 46. The method of claim 1 or 2, wherein themitochondria replaced cell has a total mtDNA copy number no greater thanabout 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about1.5 fold, or more, relative to the total mtDNA copy number of therecipient cell prior to contacting with the agent that reducesendogenous mtDNA copy number.
 47. The method of claim 1 or 2, whereinthe recipient cell is: (a) an animal cell or a plant cell, whereinanimal cell is optionally a mammalian cell, wherein the mammalian cellis optionally a somatic cell or a bone marrow cell, and wherein the bonemarrow cell is optionally a hematopoietic stem cell (HSC), or amesenchymal stem cell (MSC); (b) a cancer cell; (c) a primary cell; (d)an immune cell, wherein the immune cell is optionally selected from thegroup consisting of a T cell, a phagocyte, a microglial cell, and amacrophage, and the T cell is optionally a CD4+ T cell, a CD8+ T cell,or a chimeric antigen receptor (CAR) T cell; or (e) a senescent or nearsenescent cell. 48-58. (canceled)
 59. The method of claim 1 or 2,wherein transfer of the exogenous mitochondria and/or exogenous mtDNA isstable, wherein the exogenous mtDNA optionally alters heteroplasmy inthe recipient cell.
 60. (canceled)
 61. The method of claim 1 or 2,further comprising: (a) delivering a small molecule, a peptide, or aprotein; and/or (b) contacting the recipient cell with a second activeagent prior to co-incubating the recipient cell with exogenousmitochondria and/or exogenous mtDNA, wherein the second active agent isoptionally selected from the group consisting of large molecules, smallmolecules, or cell therapies, and the second active agent is optionallyselected from the group consisting of rapamycin, NR (NicotinamideRiboside), bezafibrate, idebenone, cysteamine bitartrate (RP103),elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone(Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrialCoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenictreatment, hypoxia, and an activator of endocytosis wherein theactivator of endocytosis is optionally a modulator of cellularmetabolism, wherein the modulator of cellular metabolism optionallycomprises nutrient starvation, a chemical inhibitor, or a smallmolecule, wherein the chemical inhibitor or the small molecule isoptionally an mTOR inhibitor, and wherein said mTOR inhibitor optionallycomprises rapamycin or a derivative thereof. 62-67. (canceled)
 68. Acomposition comprising one or more mitochondria replaced cells obtainedby the method of: (a) contacting a recipient cell with an agent thatreduces endogenous mtDNA copy number; (b) incubating the recipient cellfor a sufficient period of time for the agent to partially reduce theendogenous mtDNA copy number in the recipient cell; and (c)co-incubating (1) the recipient cell from step (b) in which theendogenous mtDNA has been partially reduced, and (2) exogenousmitochondria or exogenous mtDNA from a healthy donor, for a sufficientperiod of time to non-invasively transfer the exogenous mitochondria orthe exogenous mtDNA into the recipient cell, thereby generating amitochondria replaced cell, wherein said mitochondria replaced cellcomprises greater than 5% of exogenous mtDNA, wherein said one or moremitochondria replaced cells optionally comprise a total mtDNA copynumber no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold,about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNAcopy number of the recipient cell prior to contacting with the agentthat reduces endogenous mtDNA copy number, wherein the one or moremitochondria replaced cells optionally comprise wild-type exogenousmtDNA, wherein the exogenous mitochondria is optionally isolatedmitochondria, and the isolated mitochondria is optionally intact, andwherein the exogenous mitochondria optionally further comprisesexogenous mtDNA. 69-70. (canceled)
 71. A composition comprising an agentthat reduces endogenous mtDNA copy number, and a second active agent,wherein the composition optionally further comprises, one or morerecipient cells, exogenous mtDNA, and/or exogenous mitochondria. 72-73.(canceled)
 74. The composition of claim 68 or 71, wherein the agent thatreduces endogenous mtDNA copy number is: (a) a small molecule, whereinthe small molecule is optionally a nucleoside reverse transcriptaseinhibitor (NRTI); or (b) a fusion protein, wherein the fusion proteinoptionally comprises an endonuclease that cleaves mtDNA and amitochondrial target sequence (MTS), wherein the endonuclease optionallycleaves wild-type mtDNA, and is optionally selected from the groupconsisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc fingernuclease (ZFN), and transcription activator-like effector nuclease(TALEN), wherein the MTS optionally targets a mitochondrial matrixprotein, and the mitochondrial matrix protein is optionally selectedfrom the group consisting of cytochrome c oxidase subunit IV, cytochromec oxidase subunit VIII, and cytochrome c oxidase subunit X, and/orwherein the fusion protein is optionally transiently expressed. 75-81.(canceled)
 82. The composition of claim 68 or 71, wherein said reductionof endogenous mtDNA copy number is a partial reduction, wherein thepartial reduction is optionally a reduction of: (a) about 5% to about99% of endogenous mtDNA; (b) about 50% to about 95% of the endogenousmtDNA copy number; or (c) about 60% to about 90% of the endogenous mtDNAcopy number. 83-92. (canceled)
 93. The composition of claim 68 or 71,further comprising a second active agent, wherein the second activeagent is optionally selected from the group consisting of largemolecules, small molecules, or cell therapies, and the second activeagent is optionally selected from the group consisting of rapamycin, NR(Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate(RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176,Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone),mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin,ketogenic treatment, hypoxia, and an activator of endocytosis, whereinthe activator of endocytosis is optionally an activator of aclathrin-independent endocytosis pathway, wherein the activator ofendocytosis is optionally an activator of a clathrin-independentendocytosis pathway, wherein the clathrin-independent endocytosispathway is optionally selected from the group consisting of a CLIC/GEECendocytic pathway, Arf6-dependent endocytosis, flotillin-dependentendocytosis, macropinocytosis, circular doral ruffles, phagocytosis, andtrans-endocytosis, wherein the clathrin-independent endocytosis pathwayis optionally macropinocytosis, wherein said activator of endocytosisoptionally comprises nutrient stress, and/or an mTOR inhibitor, andwherein said mTOR inhibitor optionally comprises rapamycin or aderivative thereof. 94-100. (canceled)
 101. The composition of claim 68or 71, wherein the total mtDNA copy number of the one or moremitochondria replaced cells comprises: (a) greater than 5% of exogenousmtDNA; (b) greater than 30% of exogenous mtDNA; (c) greater than 50% ofexogenous mtDNA, or (d) greater than 75% of exogenous mtDNA. 102-106.(canceled)
 107. The composition of claim 68 or 71, wherein the exogenousmitochondria and/or exogenous mtDNA is optionally allogeneic. 108.(canceled)
 109. The composition of claim 68 or 71, wherein the one ormore cells are animal cells or plant cells, wherein the animal cells areoptionally mammalian cells, and the mammalian cells are optionallysomatic cells, and wherein the somatic cells are optionally: (a)epithelial cells, wherein the epithelial cells are thymic epithelialcells (TECs), or (b) immune cells wherein the immune cells areoptionally phagocytic cells or T cells, and the T cells are optionallyCD4+ T cells, CD8+ T cells, or chimeric antigen receptor (CAR) T cells.110-119. (canceled)
 120. The composition of claim 68 or 71, wherein theone or more mitochondria replaced cells are: (a) bone marrow cells,wherein the bone marrow cells are optionally a hematopoietic stem cell(HSC), or a mesenchymal stem cell (MSC); (b) more viable than anisogenic cell having homoplasmic endogenous mtDNA; and/or (c)efficacious in killing a cancer cell, treating an age-related disease,treating a mitochondrial disease or disorder, treating aneurodegenerative disease, treating diabetes, or a genetic disease.121-123. (canceled)
 124. The composition of claim 68 or 71, furthercomprising a small molecule, a peptide, or a protein.
 125. A compositioncomprising: (a) a senescent or near senescent cell having endogenousmitochondria; (b) isolated exogenous mitochondria from a non-senescentcell, wherein the exogenous mitochondria from the non-senescent celloptionally has enhanced function relative to the endogenousmitochondria; and (c) an agent that reduces endogenous mtDNA copynumber, wherein the agent is optionally a fusion protein, wherein thefusion protein optionally comprises an endonuclease that cleaves mtDNAand a mitochondrial target sequence (MTS), wherein the endonucleaseoptionally cleaves wild-type mtDNA, and is optionally selected from thegroup consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc fingernuclease (ZFN), and transcription activator-like effector nuclease(TALEN), wherein the MTS optionally targets a mitochondrial matrixprotein, and the mitochondrial matrix protein is optionally selectedfrom the group consisting of cytochrome c oxidase subunit IV, cytochromec oxidase subunit VIII, and cytochrome c oxidase subunit X, and/orwherein the fusion protein is optionally transiently expressed in saidsenescent or near senescent cell. 126-136. (canceled)
 137. Thecomposition of claim 125, further comprising a second active agent,wherein the second active agent is optionally selected from the groupconsisting of large molecules, small molecules, or cell therapies, andthe second active agent is optionally selected from the group consistingof rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone,cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone(RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001(alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1(Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and anactivator of endocytosis, wherein the activator of endocytosis isoptionally an activator of a clathrin-independent endocytosis pathway,wherein the activator of endocytosis is optionally an activator of aclathrin-independent endocytosis pathway, wherein theclathrin-independent endocytosis pathway is optionally selected from thegroup consisting of a CLIC/GEEC endocytic pathway, Arf6-dependentendocytosis, flotillin-dependent endocytosis, macropinocytosis, circulardoral ruffles, phagocytosis, and trans-endocytosis, wherein theclathrin-independent endocytosis pathway is optionally macropinocytosis,wherein said activator of endocytosis optionally comprises nutrientstress, and/or an mTOR inhibitor, and wherein said mTOR inhibitoroptionally comprises rapamycin or a derivative thereof. 138-143.(canceled)
 144. A pharmaceutical composition comprising an isolatedpopulation of mitochondria replaced cells having an exogenousmitochondria or an exogenous mtDNA from a healthy donor, wherein thecells are obtained by the method of claim 1 that optionally furthercomprises contacting the recipient cell with a second active agent priorto co-incubating the recipient cell with exogenous mitochondria and/orexogenous mtDNA, wherein the cells are optionally T cells orhematopoietic stem cells.
 145. (canceled)
 146. The pharmaceuticalcomposition of claim 144, further comprising (a) exogenous mitochondria,and/or (b) a pharmaceutically acceptable carrier. 147-149. (canceled)